CELL PROPAGATION AND BIOREACTOR INOCULATION
A Per.C6 cell line expressing a monoclonal anti–EpCAM (epithelial cell adhesion molecule, a cancer marker) immunoglobulin
G (IgG) antibody (licensed from Crucell) and commercially available, chemically-defined serum-free medium, CDM4 PerMab (Hyclone),
was used for the batch and perfusion phases of the cultivation. Inoculum was produced in two 1 L polycarbonate Erlenmeyer
flasks (Corning) with 300 mL culture, each incubated in a Certomat CT plus shaker incubator (Sartorius Stedim Biotech) at
85% humidity, 37 °C, 5% CO2 and a shaker rate of 240 rpm.
The bioreactor bag (CultiBag RM10 perfusion, Sartorius Stedim Biotech), equipped with single-use optical pH and DO probes,
was filled with 5 L of medium and equilibrated to process temperature. After calibration of the optical sensors, the reactor
was inoculated with a seeding density of 5 x 105 cells/mL. The initial process parameters were 19 rocks/min, a rocking angle of 6° and a temperature of 37 °C. The pH was
controlled at pH 7.1 with CO2 and 1M Na2CO3. Dissolved oxygen (DO) was controlled at 40% by automatically adjusting the gas flow rate (range 0.3 to 3 L/min) and the
composition of N2, air, and O2. For comparison to the perfusion process, a batch culture was performed using the above mentioned parameters.
CONTROL OF THE PERFUSION PROCESS
An empty bag was welded to the perfusion outlet of the bioreactor bag, and a 50 L media bag was welded to the perfusion inlet
and placed on a balance. Perfusion was started at an exchange rate of 0.5 reactor volumes/d, which corresponds to a feed and
harvest flow of 0.105 kg/h. The perfusion rate was subsequently increased based on the measured glucose and glutamine concentration
as detailed below. As the bioreactor controller automatically increased the gas flow rate and oxygen content in the process
gas, the angle and rocking rate were adjusted manually from initially 6° to 10°, and 19 rocks/min to 23 rocks/min. Due to
the increased angle and rocking rate, oxygen transfer from the headspace to the liquid phase could be further improved.
Samples were taken at regular intervals for offline pH measurement (Sartorius PB-11 pH meter) and monitored for cell count
(Nucleocounter, Sartorius Stedim Biotech), glucose, glutamine, and lactate concentration (Bioprofile 100plus Analyzer, Nova
Biomedical) and antibody concentration (ELISA).
HIGH CELL DENSITY CULTIVATION OF AN ANTIBODY EXPRESSING CELL LINE
In batch mode, a cell density of 17 × 106 cells/mL at a viability of 98% was reached (see Figure 1). This result is typical for a pH and DO controlled bioreactor
batch culture of this specific Per.C6 clone and the PerMAB medium used. Once the substrate was depleted, cells went into the
stationary phase. In contrast, in an uncontrolled process, e.g., in a shaker flask, the cell density typically reaches about
12 × 106 cells/mL under similar conditions (data not shown).
Figure 1: Viable cell density and viability of batch and perfusion process.
In perfusion culture using the BIOSTAT CultiBag RM 20, we were able to reach a very high cell density of 150 × 106 cells/mL at a viability of 99%. Up to now, the highest reported cell density of a wave-type, single-use bioreactor has been
60 × 106 cells/mL (7). Typically, we achieve about a 10-fold higher cell density in perfusion culture using this bioreactor set up
compared to the batch results (data not shown). The total cell yield was 7.5 × 1011 cells derived from 5 L of reactor culture.