2.4. Perfusion cultivations in stirred-tank bioreactors
2.4.1. XCellTM ATF-2 as cell retention device
A stirred-tank bioreactor (ez-Control, Applikon Biotechnology, The Netherlands) coupled to XCellTM ATF-2 (Repligen, USA) was set up as described previously (Alvim et al., 2019), with the exception of the hollow fiber cartridge used in the system. In the present work, a polysulfone membrane with 0.4 µm pore size and 0.005 m² of surface area (MFSL0005, Asahi Kasei, Japan) was evaluated. Cells from cell pool 1 were inoculated at approximately 0.5 × 106cells/mL in HEK TF medium and working volume was kept at 1 L ± 0.05 L. The setpoints of pH, temperature and dissolved oxygen were 7.1, 37 °C and 40 % of air saturation, respectively. A pitched-blade impeller was used for agitation, which was kept at 200 rpm during the whole cultivation. Purified air and pure oxygen were injected at a maximum combined flow rate of 0.25 L/min through a drilled hole sparger in order to maintain dissolved oxygen at the setpoint. In this run, permittivity was monitored online using a biomass sensor (FOGALE®Nanotech, France). The software accompanying the biomass sensor used a generic conversion factor for mammalian cells and provided the measured values already converted to million cells/mL.
Perfusion was started on day 4 by feeding fresh HEK TF medium supplemented with 8 mM of L-glutamine at medium exchange rates between 0.17 and 0.67 vvd. ATF recirculation rate was set initially at 0.2 L/min and then gradually increased up to 0.6 L/min, according to variations in viable cell density.
2.4.2. Inclined lamella settler as cell retention device
A stirred-tank bioreactor (RALF, Bioengineering AG, Switzerland) coupled to an inclined cell settler (model CS-10, Biotechnology Solutions, USA) was used. Gases were injected through a ring sparger, and two impellers were used (a Rushton turbine and a pitched blade impeller) to maintain agitation at 150 rpm. The cell inoculum was approximately 0.5 × 106 cells/mL, using an initial volume of 1.1 L. Setpoints of pH, temperature and dissolved oxygen were the same as in 2.4.1. Perfusion was started on day 3 and medium exchange rates varied between 0.5 and 2 vvd. Once perfusion started, the volume inside the bioreactor decreased to approximately 700 mL due to the dead volume inside the inclined settler and its recirculation loop.