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Introduction Based on the existing BIOSTAT ® CultiBag STR concept [1], Sartorius has developed a number of pivotal innovations that allow for safe and well controlled high cell density mammalian cultures in single-use stirred tank bioreactors. First-of- all a single-use exhaust cooler (plate heat exchanger) has been designed that mitigates exhaust filter clogging that might be considered a risk in high cell density cultures. Secondly, a new micro-sparger (25 holes each of a diameter of 150µm) was developed to enhance mass transfer whilst maintaining sufficient dCO2 stripping at acceptable power input and tip speeds. To test the performance limits of the BIOSTAT ® Cultibag STR equipped with the novel features, a very high cell density CHO XD ® culture was performed in the Cultibag. XD ® culture is a proprietary DSM technology that is characterized by an extended exponential growth phase resulting in a short process with extremely high viable cell densities and viabilities and a 5 – 10 fold titer boost. Equipment & Methods Equipment. A standard BIOSTAT ® CultiBag STR 50 L with jacketed vessel (Sartorius) was used to automatically control RPM, T, P, DO, pH (CO 2 and Base). Foam was controlled manually. A hollow fibre filter device was used to retain cells and product. Supervisory control and data logging was performed with the BioPAT ® MFCS/Win soſtware (Sartorius). The 50 L Cultibag STR bag was equipped with a 150 μm micro-sparger, a rushton impeller at the low and a 3-blade segment impeller at the high position, for enhanced mass transfer. Moreover a newly developed exhaust cooler was used to protect the exhaust filter from blocking by condensate or aerosols. XD ® run. The XD ® culture was performed with an IgG producing CHO clone using (only) the exponential growth phase part of a previously developed feeding protocol and bioreactor control recipe (MFCS). Medium was continuously fed using a profile. The temperature was controlled at 36.5°C, DO at 50% air saturation, pH between 7.3 and 6.8 and pCO2 below 15kPa (115 mmHg) Results Using a CHO based XD ® process, during 8 days a maximum viable cell density of almost 90 mln cells/mL at a viability >98% was achieved in the Cultibag STR. Temperature, pressure, agitation, as well as oxygen partial pressure and pH control (using disposable probes) were well maintained to set point at these very high cell densities. Due to the exhaust cooler, despite an increasing gas flow rate, no exhaust filter clogging as indicated by a pressure increase was observed at all. Moreover, even at peak viable cell density, the oxygen transfer capacity of the newly developed micro-sparger had not reached its limit. A flow rate of 1,5 LPM (0.03 vvm) pure oxygen was sufficient to maintain the DOT whilst no additional dCO 2 stripping was necessary. Conclusions The BIOSTAT ® CultibBag STR �� L equipped with the novel micro-sparger could easily cope with the oxygen demand and CO 2 removal requirements of 90 mln CHO cell/mL obtained in an XD ® process. Excellent process control was observed and no pressure build-up was detected, showing the superior security offered by a single use exhaust cooler against a standard filter heater. Acknowledgements The authors like to acknowledge the DSM Biologics R&D and PD upstream teams and in particular Foeke van der Bij, Jurjen de Jong and Xi-En Yang for carrying out the XD ® work described. References [1] Noack U, De Wilde D, Verhoeye F, Balbirnie E, Kahlert W, Adams T, Greller G, Reif O (2010). Single-use stirred tank reactor BIOSTAT CultiBag STR: Characterization and applications. In Eibl R, Eibl D (eds.), Single-Use Technology in Biopharmaceutical Manufacture: John Wiley & Sons, New Jersey High cell density XD ® cultivation of CHO cells in the BIOSTAT ® CultiBag STR 50L single use bioreactor with novel micro- sparger and single-use exhaust cooler Gerben Zijlstra 1 , Ute Noack 2 , Stefan Weisshaar 2 , Davy DeWilde 3 , Gerhard Greller 2 , Christel Fenge 2 , Rolf Douwenga 1 1/ DSM Biologics, R&D department, Rozenburglaan 17, Groningen, The Netherlands 2/.Sartorius Stedim Biotech GmbH, Göttingen, Germany 3/ Sartorius Stedim Belgium NV, Vilvoorde, Belgium, Corresponding author: [email protected] Exhaust gas cooler Gas in STR Condensate refeed Cooling liquid Exhaust- filler Exhaust Cooler �� �� �� �� �� �� �� � � � �.�� �.�� �.�� �.�� �. � �. �� K L a [1/h] aeration rate [vvm] micro sparger; �, �� mm pore size ring sparger �,� mm pore size Figure 1: Mass transfer data of the 150 μm micro-sparger compared to the standard 0.8 mm ring-sparger. Figure 2: Single use exhaust cooler to protect the exhaust filter from blocking at high aeration rates Figure 3: Picture of the 50 L Cultibag STR XD ® experiment with CHO cells Figure 5: In general excellent process control of T, p, pH, DO was observed. The optical DO and pH probes proved reliable in comparison with classical probes. No pressure build-up was observed over the exhaust filter, probably related to the exhaust cooler. Despite oxygenation with pure oxygen only, pCO 2 remained within the preset limit and no additional stripping was required. Oxygenation with 0.03 vvm was sufficient to maintain DO at setpoint. �� � � � � � � � � � � �� �� �� � � � � � � � � � � � � � Temperature °C Pressure (mbar) Current Time (days) �,�� �,�� �,�� �,�� �, �� �,�� �,�� �,�� �,�� �,�� �,�� � � � � � � � � � � pH Current Time (days) MFCS Logging not swiched on �,�� �,�� �,�� �,�� �,�� �,�� �,�� �,�� ��,� ��,� ��,� �,� �,� �,� �,� �,� � � � � � � � � � � Gasflow (L/min) pCO� (kPa) Current Time (days) Antiform depleted Airflow headspace Ocygenflow sparge pCO� p �� % ��� �� �� �� �� � � � � � � � � � � � Current Time (days) Optical probe Classical probe Optical probe Classical probe Temperature Pressure Failure Classical probe Figure 4: A viable cell density of almost 90 mln cells/mL at >98% viability was achieved in 8 days. � � � � � � � � � � ��� �� �� �� ��� �� �� �� �� �� � ���,� ��,� ��,� ��,� ��,� ��,� ��,� ��,� ��,� ��,� �,� Viable Cell Density (E6 cell/mL) Viability (%) Current Time (days) �� L XD ® VCD �� L XD ® Viability CHO 50 L Cultibag STR XD ® Run [1] Noack U, De Wilde D, Verhoeye F, Balbirnie E, Kahlert W, Adams T, Greller G, Reif O (2010). Single-use stirred tank reactor BIOSTAT CultiBag STR: Characterization and applications. In Eibl R, Eibl D (eds.), Single-Use Technology in Biopharmaceutical Manufacture: John Wiley & Sons, New Jersey XD ® is a registered trademark of DSM N.V.
