2.6 Large-scale production of RBD (319-541)-His6 in a 750-liter perfusion bioreactor

Large-scale bioreactor cultures were set up in a 750-liter bioreactor (Bioengineering, Switzerland) with a twin rotofilter system for cell retention (Bioengineering, Switzerland). The bioreactor was inoculated with 0.5 × 10⁶ cells/mL in a volume of 500 L and kept under batch culture conditions for 48 h until the cell density exceeded 2 × 10⁶ cells/mL. Perfusion was then started at a dilution rate of 0.25 vvd up to 1.8 vvd. The increase in dilution rate is related to the cell specific perfusion rate (CSPR). To maintain the CSPR within a fixed range of 0.15–0.09 nL/cell/day, which was predetermined based on the culture medium and the performance of cells in a 2-liter laboratory bioreactor, discontinuous cell bleeding was performed to establish a steady state. Culture temperature (36.5°C ± 1°C), pH (6.95 ± 0.3), and maximum stirrer speed (70 rpm) were automatically monitored and maintained. Optical density was maintained at 50% ± 30% air saturation by a constant air flow of 0.004 L/min and addition of pure oxygen as needed up to 0.006 L/min. Bioreactor cultures were sampled daily for cell concentration, viability, and supernatant analysis. The harvest, which amounted to 1,000 L, was collected continuously using a Rotofilter separator.

The material balance equations describing the growth kinetics of cultures in perfusion included a leakage factor inherent to the device used for cell retention. The cell growth rate (µ) in h^-1 was calculated using the following equation:

Where:

Dp: dilution rate in perfusion culture (h^-1),

Dc: dilution rate in continuous culture (h^-1),

α: leakage factor defined by the following equation

Where:

VCD(DRC): Density of viable cells leaking through the cell retention device (cells/mL),

VCD(B): Viable cell density in the bioreactor (cells/mL).

Finally, the specific productivity (qRBD) in pg/cell/day (pcd) was determined using the equation: