Table 1.
Summary of Parameters for Batch Production Using Adherent and Suspension-Adapted HEK293 Cells
| Cell Line | AAV293 (Adherent) |
HEKExpress (Suspension) |
||
|---|---|---|---|---|
| AAV293 1 (IGC) | AAV293 2 (IAC) | HEKExpress (IGC) | HEKExpress (IAC) | |
| Bioreactors per batch | 5 cell factories (500 cm2) | 5 cell factories (500 cm2) | 2 TubeSpin 600 | 2 TubeSpin 600 |
| Medium | 500 mL DMEM (+FBS) | 500 mL DMEM (+FBS) | 500 mL F17 | 500 mL F17 |
| Cell number at transfection | ≈500 × 106a | ≈500 × 106a | 500 × 106 | 500 × 106 |
| Cell number at harvest | 456 × 106 | 480 × 106 | 1 × 109 | 1.1 × 109 |
| Plasmid DNA per batch | 1.5 mg | 1.5 mg | 0.75 mg | 0.75 mg |
| Transfection method | calcium phosphate | calcium phosphate | PEI | PEI |
| Transfection efficiency | 83%b | 86%b | 60% | 61% |
AAV293 cells were seeded with 20 × 106 cells per cell factory (100 cm2). HEKExpress cells were seeded at 1 × 106 cells/mL. All cultures were incubated at 37°C for 72 hr. Cells in suspension were incubated with a shaking frequency of 180 rpm. Transfection efficiencies for adherent cell batches were determined by manual counting of GFP-positive cells, whereas that of cells in suspension was measured by flow cytometry.
a
Approximate cell number calculated based on the confluence of adherent cultures.
b
Determined by manual counting using a fluorescence microscope and hemocytometer.