Material and Methods
Cell Culture and Bioreactor Operation
The cell line used in this study was a Chinese hamster ovary (CHO) cell line developed for the production of a MAb. The cell
culture process was performed using chemically defined cell culture media and feeds. A typical seed train was used for the
production runs (Figure 1). After vial thawing, cells were grown in T-flasks and shake flasks for six days at 37°C, 5% CO2, and then transferred into a 2-L Cultibag RM Optical (Sartorius Stedim Biotech GbmH), followed by a 50-L wave bag. The two
last steps of the seed train were performed in bioreactors (N-2 and N-1 steps). To properly assess the performance of the
SUB as a seed train bioreactor and a production bioreactor, the cell suspension from the 50-L wave bag was split in a 50-L
SUB (Hyclone, Thermo Fisher Scientific Inc.) and a 5-L benchtop-scale glass bioreactor (BIOSTAT B-DCU Quad, Sartorius-Stedim
Biotech GbmH) for the N-2 step. The N-1 step consisted of the passage of the N-2 bioreactor, removing part of the suspension,
and adding fresh media. Then, the seed train in the 5-L glass bioreactor was used to inoculate two 5-L glass bioreactors for
the production phase. The seed train in the SUB was used to inoculate, in parallel, one SUB and two 5-L glass bioreactors
for the production phase. The experimental plan is described in Figure 1 and was performed twice.
Figure 1. Description of the process and experimental scheme. The cell amplification was performed in T-flasks and shake-flasks,
followed by a passage in a 2-L bag rocker and a 50-L bag rocker. The inoculum was splitted to inoculate the N-2 bioreactors
(SUB and 5-L glass vessel bioreactor). The N-1 SUB seed train bioreactor was used to inoculate two 5-L glass vessel bioreactors
and itself as production bioreactor with the remaining inoculum. The 5-L glass vessel seed train bioreactor was used to inoculate
one other 5-L bioreactor and itself as production bioreactor.
The process developed in the 5-L glass bioreactors was adapted for the SUB as follows: pH, temperature, DO setpoints, and
feeding strategy were unchanged compared to the 5-L bioreactor. Values for gas flow rates and agitation setpoint were scaled
up by keeping the headspace renewal rate and the power per volume constant, respectively. The pH regulation was performed
using CO2 in overlay and sodium hydroxide for the 5-L glass bioreactors.
Partial pressure of carbon dioxide (pCO2), pH, and oxygen (pO2) were controlled offline on ABL5 (Radiometer Medical). Viable cell density (VCD) and viability were measured using a Vi-Cell
automatic cell counter (Beckman Coulter). Metabolites and electrolytes were controlled offline using a Nova Bioprofile 100+
(Nova Biomedical). Protein production was determined using a Protein A–based assay on the Gyrolab platform (Gyros).
Using a Disposable DO Probe
The 50-L SUB was operated using a TruLogic RDPD (R&D and process development bioprocess) controller (Finesse Solutions). The
disposable TruFluor DO probe (Finesse Solutions) was compared to the InPro6800 polarographic probe (Mettler Toledo) at a setpoint
of 40%. The disposable DO probe was inserted in a sleeve manufactured with the SUB 50-L bag. The sleeve contained the disposable
sensor, and the probe contained a non-invasive reader connected to the transmitter. The standard DO probe was inserted with
a Kleenpack connector (Pall Corporation) after sterilization. After insertion, the standard DO probe was calibrated as usual.
The DO was controlled using the InPro6800 probe, but signals from both probes were recorded on the controller.