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Aug 1, 2013 (Vol. 33, No. 14)

Novel CHO Suspension Cell Cultivation

Studying Cellular Growth and SEAP Expression in the Finesse SmartGlass Bioreactor

  • Click Image To Enlarge +
    Figure 1. SmartGlass bioreactor together with Finesse G3Lab control system.

    A new stirred-glass bioreactor suitable for cell culture applications at benchtop scale has been designed by Finesse Solutions.

    The bioreactor with a maximum working volume of 2.0 L is controlled by the Finesse G3Lab controller representing an Intel-based microcontroller with the capability and robustness of an industrial automation solution (Figure 1). It is operated via the Finesse TruBio µC software developed on a C#/.NET foundation with state-of-the-art graphics, database management, and real-time control algorithms, which further includes preconfigured parameters for controlling of the bioprocess such as pH, DO, temperature, agitation, etc.

    This study focuses on the cultivation of CHO suspension cells in fed-batch mode using chemically defined minimal culture media and secreted human placental alkaline phosphatase (SEAP) expression, which is induced by medium exchange and temperature shift. The bioreactor was agitated by a combination of a modified Rushton turbine and a three-bladed segment impeller, which were found to be suitable for cell culture applications based on previous fluid dynamic investigations.

    The bioreactor cultivation run-up totaled 9 days and achieved maximum cell densities of 7.44 x 106 cells/mL-1 at high cell viabilities of above 96% until the end of the stationary growth phase. The maximum SEAP activity was about 63 U/mL-1. By increasing impeller speed and sparging of oxygen, it was possible to maintain the dissolved oxygen level around the set-point of 40% saturation, while preventing excessive shear stress or foam production through aeration.

  • Materials and Methods

    Overview of setting-up procedures

    Day -6: Inoculum production with CHO suspension cells (CHO XM111-10) in shake flasks.

    Day -1/0: Bioreactor and medium preparation.

    Day 0: Inoculation and starting-up of the glass bioreactor with seeding density of 0.6 x 106 cells/mL-1 in 1.0 L ChoMaster® HP-1 growth medium (that was supplemented with 2.0 g·L-1 Pluronic F-68 and 2.5 mg L-1 tetracycline).

    Day 2: Sampling, addition of 1.0 L ChoMaster HP-5 growth medium. Increase of impeller speed to maintain constant specific power inputs.

    Day 3: Sampling, sedimentation of cells and removal of supernatant for medium exchange to tetracycline-free ChoMaster HP-5 production medium.

    Day 4: Sampling, temperature shift from 37°C to 31°C.

    Days 5–9: Sampling, analytics, and harvest.

    Media

    For the seed inoculum production and the start of the cultivation in the glass bioreactor, ChoMaster HP-1 medium that was supplemented with 2.0 gL-1 Pluronic F-68 and 2.5 mgL-1 tetracycline (Cell Culture Technologies) was used. The feeding was realized with ChoMaster HP-5 growth medium, and the SEAP secretion was induced by medium exchange to tetracycline-free ChoMaster HP-5 production medium.

  • Inoculum Preparation

    The inoculum for the SmartGlass bioreactor was produced in single-use shake flask at a maximum working volume of 300 mL. The cells were inoculated with cell densities of about 0.5 x 106 cells/mL-1 and subcultivated at about 3 x 106 cells/mL-1. The flasks were shaken at a shaking rate of 120 rpm and amplitude of 25 mm. Before the seeding, fresh medium was added to the shake flasks, and the cells were allowed to settle. The supernatant was removed after about three hours, and the cells were transferred into the glass vessel.

  • Bioreactor Preparation

    The pH sensor was calibrated using pH 4.01 and pH 7.0 buffers (Mettler Toledo). The pH and DO probes were installed in the vessel and 800 mL PBS was filled into the glass vessel for sterilization (30 min, 121°C).

    The ChoMaster HP-5 growth and production media bags (FlexBoy® 3L, Sartorius Stedim Biotech) were connected to the bioreactor via LuerLock connectors in the safety cabinet. Two sterile glass bottles for media removal and antifoam solution (5 gL-1 Emulsion C, Sigma Aldrich) were connected.

    After sterilization, the PBS was replaced by 800 mL ChoMaster HP-1, and the bioreactor was connected to the control unit. Temperature and agitation control were started for sterile testing about 24 hours prior to inoculation.

    The seed was filled into the bioreactor in the sterile cabinet, and ChoMaster HP-1 was added to meet the desired initial cell density. About 30 min after the cell transfer, the first sample was taken.



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