Materials and Methods
Determination of KLa values. The KLa-values for the CultiBag RM were determined by the gassing-out method for typical rocking speeds, angles, and gas flow
rates. Ambient air and pure oxygen were used as process gasses.
Cultivation of Escherichia coli. To prepare the seed culture, E. coli BL21 (DE3), streaked out on LB agar plates, was used to inoculate two Sartorius-Stedim Biotech CultiFlask 50 disposable bioreactors
(Sartorius-Stedim Biotech DF-050MB-SSH-4), each filled with 20 mL LB medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCL).
The seed culture was grown overnight at 37 °C and 150 rpm in an incubator (Incubator Sartorius Certomat). The BIOSTAT CultiBag
RM 20 OPTICAL, part number DH-020LORM-1 using Cultibag RM 20L optical, part number DBO020L was filled with 1 L of LB medium,
preheated to 37 °C, and inoculated with the preculture to reach an optical density (OD600) of 0.15. Cultivation was started
with the following process parameters: temperature 37 °C, rocking speed 42 rpm, rocking angle 10° and airflow 0.5 lpm. Ambient
air was used for oxygen supply. Growth was monitored by measuring the optical density in regular intervals. For comparing
the growth characteristics, a baffled Erlenmeyer flask filled with 200 mL sterile LB medium was inoculated with E. coli BL21 (DE3) to a final OD600 of 0.15 and incubated at 37 °C and 150 rpm in an incubator.
Cultivation of Corynebacterium diphtheriae. The Biostat CultiBag RM 20 (Sartorius-Stedim Biotech DH-020-L-0-RM-1) was used for a feasibility study to replace a stainless-steel
reactor for seed stage fermentation in vaccine production. The CultiBag RM 20-L (Sartorius-Stedim Biotech DBO020L) was filled
with 10 L of CY-media (casamino/yeast) and inoculated with approximately 130 mL of a C. diphtheriae culture grown in an aspirator bottle to an OD590 of 8.66 resulting in an optical density (OD590) of 0.123 at the start of
fermentation. Cultivation was started using following process parameters: temperature 32 °C, rocking speed 12 rpm, rocking
angle 5.9° airflow 0.3 lpm. During the course of the cultivation, the rocking rate was raised to 42 rpm, the angle to 10°,
and the airflow to 0.55 lpm to enable high mass transfer. Ambient air was used for oxygen supply. The optical density was
measured in regular intervals throughout the process. For comparison, a stainless-steel reactor filled with 20 L of medium
was inoculated with approximately 270 mL of a C. diphtheriae culture grown in an aspirator bottle to an OD590 of 8.66 resulting in an optical density (OD590) of 0.179 at the start of
fermentation. The fermentation initial process parameters were: back pressure 5.0 psig, airflow 4.2 SLPM, and agitation 70
rpm to control the DO to ≥25%. During the fermentation, the cascade control increased the agitation to 250 rpm to maintain
Cultivation of CHO-X. A commercial CHO cell line cultivated in a serum-free fed-batch stirred-tank culture and compared with CultiBag RM using disposable
sensors for process control, referred to as the X cell line for the purposes of this paper. Temperature, pH, and dissolved
oxygen were controlled at constant, product-specific set points throughout the production cycle. The details of these cultivation
parameters are not given because of confidentiality.