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August 01, 2013 (Vol. 33, No. 14)

Novel CHO Suspension Cell Cultivation

Studying Cellular Growth and SEAP Expression in the Finesse SmartGlass Bioreactor

  • Culture Conditions

    Culture volume: 1–2 L

    Agitation speed: 140–180 rpm

    pH value: 7.2

    pH regulation: CO2 (< 0.1 slpm)

    Temperature: 37°C (growth); 31°C (protein production)

    Aeration rate: 0.1 slpm (air, headspace); 0.05–0.1 slpm (oxygen, sparger)

    Start cell density: 0.6 x 106 cells/mL-1

    Cultivation time: 9 days

  • Sampling and Analysis

    Click Image To Enlarge +
    Figure 2. (A) Total cell density and viability, (B) Concentrations of glucose and lactate, (C) SEAP activity. The arrows indicate the fresh media addition after 32 h of cultivation and the medium exchange to tetracycline-free production medium after 66 h of cultivation, respectively.

    Samples are taken in place at least twice a day by connecting a sterile 10 mL syringe via a clave adapter. in-process-control was performed by NucleoCounter NC-100 (cell density, viability; ChemoMetec), BioProfile 100 (substrate and metabolite concentrations; Nova Biomedical). Furthermore, pH value was determined by a pH meter (Mettler Toledo).

  • Results

    In Figure 2, the profiles of total cell density and viability, glucose and lactate concentrations, and SEAP activity during a cultivation time of 13 days are given. Starting from initial cell density of 0.6 x 106 cells/mL-1, the cells grew with a mean growth rate of 0.893 d-1 corresponding to a doubling time of 18.6 h.

    About 36 h after starting the cultivation, 1 L fresh growth medium was added. The growth rate after the media addition was slightly lower with 0.871 d-1 so that the total cell density prior to the medium exchange was 4.64 x 106 cells/mL-1. The maximum cell density of 7.44 x 106 cells/mL-1 was achieved after 161 h of cultivation.

    The cell viability remained high (over 96 %) until the end of the stationary phase, where all substrates were depleted. Afterward, it dropped rapidly to zero within one day, when the cultivation was stopped.

    The substrate consumption and metabolite production were comparable to our experiences with similar stirred benchtop-scale bioreactors. About 0.9 g glucose was consumed for the production of 106 cells/mL-1 and glucose was depleted after 192 h of cultivation. At the end of the exponential growth phase, the cells started to consume the lactate, whereas the maximum lactate concentration after the media exchange was 2.15 g·L-1 (Figure 2B).

  • Click Image To Enlarge +
    Figure 3. Online data of the impeller speed, dissolved oxygen level, and O2 flow rate used for the DO control.

    The SEAP activity increased rapidly after the medium exchange (Figure 2C), whereas the temperature shift (37°C to 31°C) led to an increase of the enzymatic activity. The maximum SEAP activity of 62.7 U·mL-1 was detected after about 210 h of cultivation.

  • During the complete cultivation, the DO level was maintained above critical levels aexcept of the three hours prior to the medium exchange (Figure 3). Some higher fluctuations occurred at the beginning of the cultivation, after the medium exchange, and during the stationary growth phase, which may be explained by the limited dynamic range of the mass flow controllers for oxygen and bubble attachment at the DO probes.

    However, foam formation was effectively prevented due to the low gasing rates.

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