• Authors: Napoleone A. et al.
  • Year: 2021
  • Journal: N Biotechnol.
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: Expi293F
    Description: Human embryonic kidney Fibroblast
    Known as: Expi 293-F, Expi, HEK-293 Expi


Suspension-adapted Expi293 F cells were cultured in chemically defined, serum and protein-free Expi293 Expression Medium at 37 °C in shaking incubators (80 % humidified, 125 rpm and 8% CO2). Transfection (FectoPRO) was carried out with a DNA donor to helper vector ratio of 1:3. The cell pool was established after four weeks of puromycin selection (10 μg/mL) pressure until cells showed viability of >96 %. The transfection efficiency and the establishment of the stable cell pool at that point were evaluated by their GFP expression by flow cytometry.


The transition from preclinical biological drug development into clinical trials requires an efficient upscaling process. In this context, bispecific antibody drugs are particularly challenging due to their propensity to form aggregates and generally produce low titers. Here, the upscaling process for a tetravalent bispecific antibody expressed by a piggyBac transposon-mediated stable HEK293 cell pool has been evaluated. The project was performed as a case-study at Testa Center, a non-GMP facility for scale-up testing of biologics in Sweden, and encompassed media adaptation strategies, fed-batch optimization and a novel antibody purification technology. The cell pool was adapted to different culture media for evaluation in terms of cell viability and titers compared to its original Expi293 Expression Medium. These parameters were assessed in both sequential stepwise adaption and direct media exchanges. By this, a more affordable medium was identified that did not require stepwise adaptation and with similar titers and viability as in the Expi293 Expression Medium. Fed-batch optimizations resulted in culture densities reaching up to 20 × 106 viable cells/ml with over 90% viability 12 days post-inoculum, and antibody titers three times higher than corresponding batch cultures. By implementing a novel high-speed protein A fiber technology (Fibro PrismA) with a capture residence time of only 7.5 seconds, 8 L of supernatant could be purified in 4.5 h without compromising the purity, structural integrity and function of the bispecific antibody. Results from this study related to medium adaptation and design of fed-batch protocols will be highly beneficial during the forthcoming scale-up of this therapeutic antibody.

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