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  • 1.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Tördahl, Karin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Perroud, Philip
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Unthan, Simon
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Schmidt, Johannes RM
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Doverskog, Magnus
    IMED, AB, Solna, Sweden.
    Study of Alternating Tangential Flow filtration for perfusion and harvest in Chinese Hamster Ovary cells cultivation2010In: Proceedings of the Cell Culture Engineering Conference XII, April 25-30, 2010, Banff, Canada, 2010Conference paper (Other academic)
    Abstract [en]

    Perfusion is a mode of operation where a continuous replacement of the conditioned medium by fresh medium is operated. It has the advantage of allowing high cell densities. This mode of operations is also required for some instable proteins since the cell-free supernatant containing the product of interest is immediately stored at low temperature where the proteolysis is not active. The ATF filtration device, Alternating Tangential Flow, has been designed to perfuse mammalian cell cultivation process and is used (or studied) nowadays for applications like perfusion, medium renewal, harvest, etc. The cell broth circulation back and forward in the filter prevents the filter clogging and the design ensures a low shear not damageable for the cells.

    A perfusion process operated by ATF filtration and using CHO cells producing a monoclonal antibody was developed in a 2 L bioreactor. The medium did not contain animal derived components. Cell densities above 40 x 1E6 cells/mL were obtained with a perfusion rate of 2 reactor volume/day. The highest cell density observed was 48 x 1E6 cells/mL. These high cell densities were challenging for the aeration. Pure oxygen aeration by large bubbles from an open tube resulted in satisfying oxygenation until 25 to 30 x 1E6 cells/mL but became limiting at higher cell densities due to the low kLa of these bubbles and the small liquid height. At higher cell densities, a porous sparger with pure oxygen was used either alone or in combination with the open tube aeration. Automatic delivery of antifoam C and pluronic counteracted the effect of small bubble foam deleterious for the cells. From an operation point-of-view, the perfusion operated by the ATF device was satisfying, without filter fouling, easy to operate and to adjust in comparison with other separation devices by filtration or gravitation.

    Finally harvesting by ATF filtration was evaluated in comparison with ‘one-way’ tangential flow filtration, TFF, on an IgG producing CHO fed-batch process produced in 2 L bioreactor and in 20 L bioreactor. Different settings and filter sizes were compared and the effect of varying the flow rate was studied.

  • 2.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Tördal, Karin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Perroud, Philip
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Unthan, Simon
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Schmidt, Johannes R.M.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Doverskog, Magnus
    IMED, AB, Solna, Sweden.
    Study of Alternating Tangential Flow filtration for perfusion and harvest in Chinese Hamster Ovary cells cultivation2010Other (Other academic)
    Abstract [en]

    Perfusion is a mode of operation where a continuous replacement of the conditioned medium by fresh medium is operated. It has the advantage of allowing high cell densities. This mode of operations is also required for some instable proteins since the cell-free supernatant containing the product of interest is immediately stored at low temperature where the proteolysis is not active. The ATF filtration device, Alternating Tangential Flow, has been designed to perfuse mammalian cell cultivation process and is used (or studied) nowadays for applications like perfusion, medium renewal, harvest, etc. The cell broth circulation back and forward in the filter prevents the filter clogging and the design ensures a low shear not damageable for the cells.

    A perfusion process operated by ATF filtration and using CHO cells producing a monoclonal antibody was developed in a 2 L bioreactor. The medium did not contain animal derived components. Cell densities above 40 x 106 cells/mL were obtained with a perfusion rate of 2 reactor volume/day. The highest cell density observed was 48 x 106 cells/mL. These high cell densities were challenging for the aeration. Pure oxygen aeration by large bubbles from an open tube resulted in satisfying oxygenation until 25 to 30 x 106 cells/mL but became limiting at higher cell densities due to the low kLa of these bubbles and the small liquid height. At higher cell densities, a porous sparger with pure oxygen was used either alone or in combination with the open tube aeration. Automatic delivery of antifoam C and pluronic counteracted the effect of small bubble foam deleterious for the cells. From an operation point-of-view, the perfusion operated by the ATF device was satisfying, without filter fouling, easy to operate and to adjust in comparison with other separation devices by filtration or acceleration.

     

    Finally harvesting by ATF filtration was evaluated in comparison with ‘one-way’ tangential flow filtration, TFF, on an IgG producing CHO fed-batch process produced in 2 L bioreactor. In both operation modes, ATF and TFF, filter fouling occurred after several minutes and the total process time was comparable but an important difference was that the viability drop obtained when using ATF was 15 % while it was 45 % using the TFF.

  • 3.
    Doverskog, Magnus
    et al.
    KTH, Superseded Departments, Biotechnology.
    Bertram, Eva.
    KTH, Superseded Departments, Biotechnology.
    Ljunggren, Jan
    Öhman, Lars
    Sennerstam, Roland
    Häggström, Lena
    KTH, Superseded Departments, Biotechnology.
    Cell cycle progression in serum-free cultures of Sf9 insect cells: Modulation by conditioned medium factors and implications for proliferation and productivity2000In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 16, no 5, p. 837-846Article in journal (Refereed)
    Abstract [en]

    Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterized by an accumulation of 50-70% of the cells in the G(2)/M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G(1) and in particular into the S phase. Maximum rate of proliferation (mu(N,max)) was reached 24-48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G(2)/M phase. The following declining mu(N) could be explained by a slow increase in the G(2)/M cell population. However, at approximately 100 h, an abrupt increase in the amount of G(2)/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G(2)/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase mu(N,max), decrease the time to reach mu(N,max), and decrease the synchronization of cells in G(2)/M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of beta-galactosidase up to 68-75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean;population cell volume had attained a minimum, and this occurred 24 h before the switch into the G(2)/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G(2)/M cell cycle arrest.

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