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  • 1.
    Al-Khalili, Lubna
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Gillner, Karin
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Åstrand, Carolina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Shokri, Atefeh
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Hughes-Brittain, Nanaaya
    McKean, Robert
    Robb, Brendan
    Chotteau, Véronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Characterization of Human CD133+Cells in Biocompatible Poly(l-lactic acid) Electrospun Nano-Fiber Scaffolds2016In: Journal of Biomaterials and Tissue Engineering, ISSN 2157-9083, E-ISSN 2157-9091, Vol. 6, no 12, 959-966 p.Article in journal (Refereed)
    Abstract [en]

    CD133+ cells are potential myogenic progenitors for skeletal muscle regeneration to treat muscular dystrophies. The proliferation of human CD133+ stem cells was studied for 14 days in 3D biomimetic electrospun poly-L-lactic acid (PLLA) nano-fiber scaffolds. Additionally, the myogenic differentiation of the cells was studied during the last 7 days of the culture period. The cells were homogeneously distributed in the 3D scaffolds while colony formation and myotube formation occurred in 2D. After a lag phase due to lower initial cell attachment and an adaptation period, the cell growth rate in 3D was comparable to 2D after 7 and 14 days of culture. The expression of the stem cell (SC) marker PAX7 was 1.5-fold higher in 3D than 2D while the differentiation markers MyoG, Desmin and MyoD were only slightly changed (or remain unchanged) in 3D but strongly increased in 2D (12.6, 3.9, and 7.9-fold), and the myotube formation observed in 2D was absent in 3D. The marker expression during proliferation and differentiation, together with the absence of myotubes in 3D, indicates a better maintenance of stemness in 3D PLLA and stronger tendency for spontaneous differentiation in 2D culture. This makes 3D PLLA a promising biomaterial for the expansion of functional CD133+ cells.

  • 2. Castan, Andreas
    et al.
    Fäldt, Erik
    Mölleryd, Carin
    Lindskog, Eva
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Cultivation process intensification utilizing a WAVE Bioreactor (TM) for perfusion cell cultures2013In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 245Article in journal (Other academic)
  • 3.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Fed-batch or perfusion for the production of biopharmaceuticals by animal cell cultivation?2009Other (Other academic)
    Abstract [en]

    Fed-batch or perfusion modes are today’s options for the cultivation process development of new candidates drugs. In the cases of unstable proteins, perfusion is the obligatory choice. Otherwise, this choice is dictated by the need of simplicity, the existing technical know-how in the company and the available equipment (e.g. bioreactor size, perfusion device, etc.). The higher risk of contamination and higher technical challenge of perfusion processes lead often to the selection of fed-batch for commercial processes. This is reinforced today in the case of antibody production in established biotech companies by the use of generic fed-batch processes with high yield production and low accumulation of toxic by-products (e.g. lactate, ammonia). However perfusion can be a valuable option due to the usage of smaller bioreactors, today’s availability of scalable low shear force perfusion system (e.g. ATF) and potentially the lower requirement of process development for perfusion. Perfusion can therefore be an attractive choice for instance for the production of glycoproteins, which are not antibody, and/or for small companies, which do not have generic process.

  • 4.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Mastering Oxygen and Carbon Dioxide in Cell Culture2010Other (Other academic)
    Abstract [en]

    Mammalian cells need adequate aeration for the cellular respiration and simultaneously produce carbon dioxide. Aeration in pilot and large scale cultivation is operated by blowing oxygen or oxygen enriched air in the culture, i.e. sparging. The gas bubbles reaching the surface generate foam formation, which is well known to be damageable for the cells. Therefore, it can be preferable to reduce the bubble sparging while maintaining the oxygen concentration in the culture. Carbon dioxide produced by the cells can accumulate in the culture in case its removal rate from the liquid phase is not fast enough. This provokes an acidification of the medium, which has to be compensated by alkali addition. High levels of carbon dioxide and high levels of alkali can be damageable for the process performances. A common way to actively remove the carbon dioxide from the liquid phase is to benefit from the bubbles moving to the liquid surface. Obviously, large scale cultivation can present a dilemma in which reduced sparging is favoured for the aeration strategy in order to avoid cell damage by excessive foaming while increased sparging provide reduced accumulation of carbon dioxide. Typical approaches used in the biopharmaceutical industry for aeration and carbon dioxide removal will be reviewed in the presentation. 

  • 5.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Perfusion Processes2015In: Animal Cell Culture / [ed] Mohamed Al-Rubeai, Switzerland Springer: Springer , 2015, 407-443 p.Chapter in book (Refereed)
    Abstract [en]

    The interest for perfusion is increasing nowadays. This new focus has emerged from a synergy of a demand for disposable equipment and the availability of robust cell separation device, as well as the need for higher flexibility and lower investment cost. The cell separation devices mostly used today are based on filtration, i.e. alternating flow filtration, tangential flow filtration, spin-filter, or acceleration/gravity, i.e. inclined settler, centrifuge, acoustic settler. This paper gives an introduction to the basic concepts of perfusion and its practical implementation. It reviews the actual cell separation devices and describes the approaches used in the field to develop and optimize the perfusion processes.

  • 6.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Diana, Rafael
    Ge Healthcare Bio-sciences Ab.
    Kaisermayer, Christian
    Ge Healthcare Bio-sciences Ab.
    Lindskog, Eva
    Ge Healthcare Bio-sciences Ab.
    Robinson, Craig
    Ge Healthcare Bio-sciences Ab.
    Rucker, Jimmie L.
    Ge Healthcare Bio-sciences Ab.
    Walsh, Kieron D.
    Ge Healthcare Bio-sciences Ab.
    Flexible bag for cultivation of cells2011Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    An inflatable bioreactor bag for cell cultivation, which comprising a top and a bottom sheet of flexible material, joined together to form two end edges and two side edges, wherein one baffle or a plurality of baffles extend from the bottom sheet in a region where the shortest distance to any one of the two end edges is higher than about one fourth of the shortest distance between the two end edges.

  • 7.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Hjalmarsson, Håkan
    KTH, School of Electrical Engineering (EES), Automatic Control.
    Tuning of dissolved oxygen and pH PID control parameters in large scale bioreactor by lag control2008In: Proceedings of the Cell Culture Engineering XI Conference, 2008Conference paper (Refereed)
    Abstract [en]

    Achieving satisfying DO and pH controllers are often challenges for pilot and large scale mammalian cultivation. Unsatisfactory DO or pH controls can imply fatal effects for the culture. Large scale bioreactors have long response times due to long mixing times compared to small scale systems where control tuning of DO and pH is not so challenging.

    A method was developed to tune the DO controller PID parameters of a 50 L bioreactor (wv) controlled by a continuous oxygen flow of microbubbles. DO control by continuous flow of pure oxygen microbubbles can oscillate quite widely showing instable behaviour. The method, called lag control here, was based on a lead lag control design by Bode analysis where the prediction part, i.e. ‘lead’ part was omitted. A comparison of this method with a pole placement approach showed the advantage of the lag control. It was decided to omit the derivate part which could lead to instability caused by the long delay observed between the applied oxygen flow and the response detected by the DO probe. Applying the lag control method resulted in a highly satisfactory DO control. In this system, the oxygen microbubbles were almost completely consumed before reaching the liquid surface as demonstrated by the absence of foam. So the oxygen flow used to maintain the DO gave an excellent indication of the cellular oxygen consumption. The control system was robust against all the perturbations of this system, i.e. cell growth, cell bleed, addition of air-saturated fresh medium, DO set point change and a second gas sparger used to strip out the carbon dioxide. The method was first tested with the sulphite oxidation method simulating the oxygen consumption with copper as catalyst to establish the PID parameters. Then the selected parameters were successfully used during cell cultivation. Following this, an adaptation of the method was done in order to avoid the sulphite oxidation method, which leaves copper traces in the bioreactor. This was successfully used in a 400 L bioreactor (wv) for the DO controller by continuous oxygen flow of microbbubles. The lag controller method was finally modified to tune the pH controller of the same 400 L bioreactor with control upward by alkali addition or downwards by pulsed carbon dioxide addition.

  • 8.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Jiang, Yun
    Biovitrum/SOBI, Sweden.
    westin, Jeanette
    Biovitrum/SOBI, Sweden.
    Dahlenborg, K
    Biovitrum/SOBI, Sweden.
    Sjöblom-Hallén, A
    Biovitrum/SOBI, Sweden.
    Svensson, Erik
    Biovitrum/SOBI, Sweden.
    Öberg, Mikael
    Biovitrum/SOBI, Sweden.
    Development of a fed-batch process for the production of a recombinant protein X in CHO-GS system: Case study from the cell to reactor process ready for pilot scale cultivation2010In: Cells and Culture: Proceedings of the 20th ESACT / [ed] Noll T, Springer Science+Business Media B.V., 2010, 723-725 p.Conference paper (Other academic)
    Abstract [en]

    A new cell line was created using CHO-GS system. The most promising clones were adapted to different base cultivation media leading to the selection of one medium. The fed-batch process development was performed in spinner, shake flask and bioreactor scale. It included the selection of a feed medium, the choice of the feed strategy and the optimisation of the glucose feeding. The process was then simplified by using a single feed including the feed medium and the glucose feed. Finally up-scaling parameters like aeration and CO2 stripping were studied in 3 L and 15 L bioreactors in preparation for pilot scale operation. This process proved to be robust, reproducible and suitable for large and commercial scale operation.

  • 9.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Lindqvist, Anna
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Study of the effect of high pH and alkali addition in a cultivation of Chinese Hamster Ovary cell2012In: Proceedings of the 21st Annual Meeting of the European Society for Animal Cell Technology (ESACT) / [ed] Jenkins, Nigel; Barron, Niall; Alves, Paula, Springer Science+Business Media B.V., 2012, 323-326 p.Conference paper (Refereed)
    Abstract [en]

    This work aimed at studying the impact of alkali addition in a Chinese Hamster Ovary cell culture. Two phenomena were studied, the kinetic rate of direct cell death in presence of high pH and the effect of transitory single contact of high pH on cell viability and growth. Contact with pH 11 or 10 did not provoke immediate cell lysis. The cells survived several minutes to such conditions. Contact with pH 11 during 2 minutes, with pH 10 during 5 minutes, with pH 9 during 5 minutes or 10 minutes did not affect the viability. In these conditions, the growth was not affected except after 5 minutes contact at pH 10 or 10 minutes contact at pH 9 for which the growth was slowed down the first day only. As expected, NaOH addition affected the cells more than Na2CO3 addition. This was due to a higher pH but could be even observed at the same pH (10).

  • 10.
    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.
    Study of a perfusion process of Chinese Hamster Ovary cells by ATF filtration in bioreactor2009Other (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 has been designed to perfuse mammalian cell cultivation process. 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.

    The purpose of the present work was to develop and study a perfusion process of Chinese Hamster Ovary cells producing a monoclonal antibody by ATF filtration in a 2 L working volume bioreactor. A serum-free medium was used (Irvine Scientific IS CHO-CD). Cell densities above 40 x 106 cells/mL were obtained. 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. The performances of the perfusion by ATF filtration, the aeration and the use of anti-foam are presented and discussed.

  • 11.
    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.

  • 12.
    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.

  • 13.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Wang, Jingjiao
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Tolf, Erika
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Wicklund, Linn
    Karolinska Institute, Department of Neurobiology, Care Sciences and Society.
    Hovatta, Outi
    Karolinska Institutet, Department of Clinical Science.
    Marutle, Amelia
    Karolinska Institute, Department of Neurobiology, Care Sciences and Society.
    Comparison of cultivation in Techne spinner, Bellco spinner, shake flask and T-flask of human embryonic stem cells2010In: Proceedings of the SBE's Second International Conference on Stem Cell Engineering, 2010Conference paper (Other academic)
    Abstract [en]

    The recent progress in regenerative medicine indicates that pluripotent human embryonic stem cells (hESCs) may hold great potential providing cellular models for drug development and screening, modelling diseases as well as aid in the development of future cell-based therapies for neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease. Crucial to the success of generating specialized cell populations, is an understanding of the mechanisms, which influence the control of cell growth and differentiation by extrinsic and intrinsic factors. Nowadays, a limitation for the use of hESCs is the lack of proliferation methods in large scale. The purpose of the present work was to study several cultivation systems, which could potentially provide large-scale cultivation processes suitable for human therapy applications. Pluripotent human embryonic stem cells (hESCs), isolated from the inner cell mass of the blastocyst, were cultivated undifferentiated as embryoids bodies, i.e. large spherical aggregates of cells, in absence of serum and feeder layer. The cell growth and culture behavior in T-falsk, orbitally agitated shake flask, Bellco stirred spinner and Techne stirred spinner were observed. In Bellco spinner, the cells were agitated by a rotating impeller providing a movement comparable to stirred bioreactors. In Techne spinner, a slow and gentle orbital movement provided by a rotating bulb-ended stirrer maintained the cells in suspension. The design of this latter spinner allowed lower shear stress in comparison to Bellco spinner and shake flask. It was observed that the cell growth was fastest in Techne spinner followed by cultivation in T-flask and then cultivation in shake flask. Cultivating in Bellco spinner resulted in embryoid dissociation and viability decrease after 14 days. A larger number of single cells, i.e. cells not growing in aggregates, was observed in the static T-flask culture compared to the agitated systems, i.e. shake flask, Bellco spinner or Techne spinner. Probably the agitation promoted the spontaneous aggregation of the cells in spheres. In particular the Techne spinner allowed the most perfect spherical form among the different compared systems. Finally it was observed that hypoxia with 4 % oxygen concentration improved significantly the growth in Techne spinner or T-flask in comparison with normoxia with 21 % oxygen concentration. It was concluded that cultivation in Techne spinner under hypoxia was the most favorable condition among the ones studied here. The agitation provided by Techne spinner improved the cell growth in comparison with static system (T-flask). However using the other agitated systems, shake flask and Bellco spinner, was not comparably beneficial to the cell growth and viability, probably due to the higher shear stress of these systems compared to Techne spinner.

  • 14.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO).
    Wåhlgren, Caroline
    Biovitrum/SOBI, Sweden.
    Jiang, Yun
    Biovitrum/SOBI, Sweden.
    Svensson, Erik
    Biovitrum/SOBI, Sweden.
    Process for cultivating animal cells comprising the feeding of plant-derived peptones2005Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    A process for cultivating animal cells producing complex proteins, wherein one plant-derived peptone or a combination of plant-derived peptones is fed to the cell culture, as well as a method for reducing the toxic effect of over-feeding amino acids during a fed-batch process for cultivating animal cells producing complex proteins.

  • 15.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Wåhlgren, Caroline
    Biovitrum/SOBI, Sweden.
    Pettersson, Helena
    Biovitrum/SOBI, Sweden.
    Effect of Peptones and Study of Feeding Strategies in a CHO Based Fed-batch Process for the Production of a Human Monoclonal Antibody2007In: Cell Technology for Cell Products: Proceedings of the 19th ESACT Meeting, Harrogate, UK, June 5-8, 2005 / [ed] Smith R, Dordrecht, The Netherlands: Springer Netherlands, 2007, 371-374 p.Conference paper (Other academic)
    Abstract [en]

    Eight commercial peptones, derived from plants, were studied for their ability of improving the cell growth and the productivity of a CHO cell line producing a human monoclonal antibody. They were also compared to yeast, lactalbumin and meat derived peptones. Seven plant peptones were selected and further studied in combination by Design of Experiment. The best three peptones were then tested in combinations in fed-batch cultivation. The fed-batch process was based on low concentrations of glucose and glutamine with feeding of amino acids, peptones and feed medium including vitamins, metal traces and biosynthesis precursors. This process was based on Biovitrum protein-free proprietary medium for the base medium and the feeding medium. Different feeding strategies, different peptone combinations and phosphate feeding were studied for their ability to improve the cell density, the cell specific productivity and the cultivation longevity

  • 16.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Clincke, Marie-Francoise
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Very High Cell Density in Perfusion of CHO Cells by ATF, TFF, Wave Bioreactor, and/or CellTank Technologies: Impact of Cell Density and Applications2014In: Continuous Processing in Pharmaceutical Manufacturing / [ed] Ganapathy Subramanian, Germany Weinheim: Wiley-VCH Verlagsgesellschaft, 2014, 339-356 p.Chapter in book (Other academic)
  • 17.
    Chotteau, Véronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Hjalmarsson, Håkan
    KTH, School of Electrical Engineering (EES), Automatic Control. KTH, School of Electrical Engineering (EES), Centres, ACCESS Linnaeus Centre.
    Tuning of dissolved oxygen and pH PID control parameters in large scale bioreactor by lag control2012In: Proceedings of the 21st Annual Meeting of the European Society for Animal Cell Technology (ESACT), 2012, 327-330 p.Conference paper (Refereed)
    Abstract [en]

    A method has been developed to tune the DO and pH controller PID parameters for pilot / large scale mammalian cultivation. Our approach is to identify a model of the variable to be controlled (e.g. DO, pH) and to design several possible PID controllers based on this model. The controllers were first tested in computer simulations, followed by wet simulation and finally the best controller was tested on the real process. The approach is developed for the tuning of the DO controller of a 50 L bioreactor using microbubble continuous oxygen flow. The method, called lag control here, is based on a lead lag control design using Bode analysis where the prediction part is omitted. Experiments show that the approach results in a highly satisfactory DO control. The oxygen microbubbles were almost completely consumed before reaching the liquid surface so the oxygen flow used to maintain the DO gave an excellent indication of the cellular oxygen consumption. The control system was robust against all the perturbations, i.e. cell growth, cell bleed, addition of air-saturated fresh medium, DO set point change and a second gas sparger used to strip out the carbon dioxide. This approach was also successfully used for the tuning of a 400 L bioreactor DO controller and pH controller.

  • 18.
    Clincke, Marie-Francoise
    et al.
    KTH, School of Biotechnology (BIO).
    Mölleryd, Carin
    KTH, School of Biotechnology (BIO).
    Samani, Puneeth K.
    KTH, School of Biotechnology (BIO).
    Lindskog, Eva
    Fäldt, Eric
    Walsh, Kieron
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE bioreactorpart II: Applications for antibody production and cryopreservation2013In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 29, no 3, 768-777 p.Article in journal (Refereed)
    Abstract [en]

    A high cell density perfusion process of monoclonal antibody (MAb) producing Chinese hamster ovary (CHO) cells was developed in disposable WAVE Bioreactor using external hollow fiber (HF) filter as cell separation device. Tangential flow filtration (TFF) and alternating tangential flow (ATF) systems were compared and process applications of high cell density perfusion were studied here: MAb production and cryopreservation. Operations by perfusion using microfiltration (MF) or ultrafiltration (UF) with ATF or TFF and by fed-batch were compared. Cell densities higher than 108 cells/mL were obtained using UF TFF or UF ATF. The cells produced comparable amounts of MAb in perfusion by ATF or TFF, MF or UF. MAbs were partially retained by the MF using ATF or TFF but more severely using TFF. Consequently, MAbs were lost when cell broth was discarded from the bioreactor in the daily bleeds. The MAb cell-specific productivity was comparable at cell densities up to 1.3 x 108 cells/mL in perfusion and was comparable or lower in fed-batch. After 12 days, six times more MAbs were harvested using perfusion by ATF or TFF with MF or UF, compared to fed-batch and 28x more in a 1-month perfusion at 108 cells/mL density. Pumping at a recirculation rate up to 2.75 L/min did not damage the cells with the present TFF settings with HF short circuited. Cell cryopreservation at 0.5 x 108 and 108 cells/mL was performed using cells from a perfusion run at 108 cells/mL density. Cell resuscitation was very successful, showing that this system was a reliable process for cell bank manufacturing.

  • 19.
    Clincke, Marie-Francoise
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Mölleryd, Carin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Samani, Puneeth K
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Lindskog, Eva
    GE Healthcare, Uppsala, Sweden.
    Walsh, Kieron
    GE Healthcare, Westborough, MA, USA.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Perfusion of an IgG producing CHO cell line at very high cell density by ATF or by TFF in WAVE Bioreactor™2011Conference paper (Other academic)
    Abstract [en]

    Perfusion of an IgG producing CHO cell line was performed in a WAVE Bioreactor™ using either Alternating Tangential Flow or Tangential Flow Filtration. The properties and performances obtained with both filtration systems were compared. Very high cell densities were achieved and could be stably maintained. Then the cell density could be significantly further increased showing the capacity of the system set-up.

  • 20.
    Clincke, Marie-Francoise
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Mölleryd, Carin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Samani, Puneeth K.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Lindskog, Eva
    GE Healthcare, Uppsala, Sweden.
    Walsh, Kieron
    GE Healthcare, Westborough, MA, USA.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Perfusion of an IgG producing CHO cell line at very high cell density by ATF or by TFF in WAVE Bioreactor™2011Conference paper (Other academic)
    Abstract [en]

    Perfusion of an IgG producing CHO cell line was performed in a WAVE Bioreactor™ using either Alternating Tangential Flow or Tangential Flow Filtration. The properties and performances obtained in this bioreactor with both filtration systems were studied.

    • Very high cell densities were achieved and could be stably maintained at high viability indicating of a healthy process suitable for instance for efficient cell banking.

    Cell density could be significantly further increased showing the capacity of the system set-up.

  • 21.
    Clincke, Marie-Francoise
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Mölleryd, Carin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Lindskog, Eva
    GE Healthcare, Uppsala, Sweden.
    Walsh, Kieron
    GE Healthcare, Westborough, MA, USA.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Study of a recombinant CHO cell line producing a monoclonal antibody by ATF or TFF external filter perfusion in a WAVE Bioreactor™2011In: BMC Proceedings, 2011, Volume 5, Supplement 8, P105, BioMed Central, 2011, 105- p.Conference paper (Refereed)
    Abstract [en]

    Major advantages of perfusion are high cell numbers and high total production in a relatively small size bioreactor. Moreover, perfusion is optimal when the product of interest is unstable or if the product yield is low. On the other hand, disadvantages are for example technical challenges originating from non-robust cell separation devices as well as sterility concerns from the more complex set-up needed.

    In the present work, the use of a WAVE Bioreactor™ system 20/50 in perfusion mode with10 L disposable Cellbag™ bioreactors customized with two dip tubes in combination with disposable hollow fiber filters as external cell separating devices were investigated. A comparison between Alternating Tangential Flow (ATF) and Tangential Flow Filtration (TFF) was performed using a recombinant CHO cell line producing a monoclonal antibody (mAb) as a model system. 

  • 22.
    Clincke, Marie-Francoise
    et al.
    KTH, School of Biotechnology (BIO).
    Mölleryd, Carin
    KTH, School of Biotechnology (BIO).
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Lindskog, Eva
    Walsh, Kieron
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor. Part I. Effect of the cell density on the process2013In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 29, no 3, 754-767 p.Article in journal (Refereed)
    Abstract [en]

    High cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor using external hollow fiber filter as cell separation device. Both classical tangential flow filtration (TFF) and alternating tangential flow system (ATF) equipment were used and compared. Consistency of both TFF- and ATF-based cultures was shown at 20-35 x 106 cells/mL density stabilized by cell bleeds. To minimize the nutrients deprivation and by-product accumulation, a perfusion rate correlated to the cell density was applied. The cells were maintained by cell bleeds at density 0.9-1.3 x 108 cells/mL in growing state and at high viability for more than 2 weeks. Finally, with the present settings, maximal cell densities of 2.14 x 108 cells/mL, achieved for the first time in a wave-induced bioreactor, and 1.32 x 108 cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell density was limited by the membrane capacity for the encountered high viscosity and by the pCO2 level. Using ATF, the cell density was limited by the vacuum capacity failing to pull the highly viscous fluid. Thus, the TFF system allowed reaching higher cell densities. The TFF inlet pressure was highly correlated to the viscosity leading to the development of a model of this pressure, which is a useful tool for hollow fiber design of TFF and ATF. At very high cell density, the viscosity introduced physical limitations. This led us to recommend cell densities under 1.46 x 108 cell/mL based on the analysis of the theoretical distance between the cells for the present cell line.

  • 23.
    Hagrot, Erika
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Oddsdóttir, Hildur Aesa
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Hosta, Joan Gonzalez
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Jacobsen, Elling W.
    KTH, School of Electrical Engineering (EES), Automatic Control.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Poly-pathway model, a novel approach to simulate multiple metabolic states by reaction network-based model - Application to amino acid depletion in CHO cell culture2016In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 228, 37-49 p.Article in journal (Refereed)
    Abstract [en]

    Mammalian cell lines are characterized by a complex and flexible metabolism. A single model that could describe the variations in metabolic behavior triggered by variations in the culture conditions would be a precious tool in bioprocess development. In this paper, we introduce an approach to generate a poly-pathway model and use it to simulate diverse metabolic states triggered in response to removal, reduction or doubling of amino acids in the culture medium of an antibody-producing CHO cell line. Macro-reactions were obtained from a metabolic network via elementary flux mode enumeration and the fluxes were modeled by kinetic equations with saturation and inhibition effects from external medium components. Importantly, one set of kinetic parameters was estimated using experimental data of the multiple metabolic states. A good fit between the model and the data was obtained for the majority of the metabolites and the experimentally observed flux variations. We find that the poly-pathway modeling approach is promising for the simulation of multiple metabolic states.

  • 24.
    Hagrot, Erika
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Oddsdóttir, Hildur Æsa
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Hosta, Joan Gonzalez
    Jacobsen, Elling W.
    KTH, School of Electrical Engineering (EES), Automatic Control.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Poly-pathway model, a novel approach to simulate multiple metabolic states by reaction network-based model - Application to CHO cell cultureManuscript (preprint) (Other academic)
  • 25.
    Jiang, Yun
    et al.
    Biovitrum/SOBI, Sweden.
    Svensson, Erik
    Biovitrum/SOBI, Sweden.
    Chotteau, Veronique
    Improvement of a CHO Fed-Batch Process by Fortifying with Plant Peptones2010In: Cells and Culture: ESACT Proceedings, 2010, Volume 4, Part 3 / [ed] Noll T, Springer, 2010, 281-284 p.Conference paper (Other academic)
    Abstract [en]

    A serum-free fed-batch process was developed for production of a human monoclonal antibody in Chinese hamster ovary (CHO) cells based on Biovitrum’s proprietary low protein serum-free medium without animal derived components (BVT4). The cells were fed with glucose, glutamine and Biovitrum’s proprietary low protein serum-free feed medium without animal derived components enriched with amino acids, vitamins, metal traces, peptones, and biosynthesis precursors. To improve the performance of the fed-batch process, we developed the use of plant peptones by studying the dose and timing of the peptone feeding. Different doses of peptone cocktail and amino acid cocktail, as well as different combinations of pep- tone and amino acid cocktails were first screened in 50 ml filter tubes on an AgCell shaker table. The best combinations were then assessed in spinner and 3 L bioreactor cultures. To reinforce our findings, the antibody-producing CHO cells were adapted to a disclosed serum-free medium DMEM/F12 and the beneficial effects of pep- tones were confirmed in a fed-batch process based on the DMEM/F12 serum-free medium.

  • 26.
    Oddsdottir, Hildur Aesa
    et al.
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Hagrot, Erika
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Forsgren, Anders
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Robustness analysis of elementary flux modes generated by column generation2016In: Mathematical Biosciences, ISSN 0025-5564, E-ISSN 1879-3134, Vol. 273, 45-56 p.Article in journal (Refereed)
    Abstract [en]

    Elementary flux modes (EFMs) are vectors defined from a metabolic reaction network, giving the connections between substrates and products. EFMs-based metabolic flux analysis (MFA) estimates the flux over each EFM from external flux measurements through least-squares data fitting. The measurements used in the data fitting are subject to errors. A robust optimization problem includes information on errors and gives a way to examine the sensitivity of the solution of the EFMs-based MFA to these errors. In general, formulating a robust optimization problem may make the problem significantly harder. We show that in the case of the EFMs-based MFA, when the errors are only in measurements and bounded by an interval, the robust problem can be stated as a convex quadratic programming (QP) problem. We have previously shown how the data fitting problem may be solved in a column-generation framework. In this paper, we show how column generation may be applied also to the robust problem, thereby avoiding explicit enumeration of EFMs. Furthermore, the option to indicate intervals on metabolites that are not measured is introduced in this column generation framework. The robustness of the data is evaluated in a case-study, which indicates that the solutions of our non-robust problems are in fact near-optimal also when robustness is considered, implying that the errors in measurement do not have a large impact on the optimal solution. Furthermore, we showed that the addition of intervals on unmeasured metabolites resulted in a change in the optimal solution.

  • 27.
    Oddsdóttir, Hildur Æsa
    et al.
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Hagrot, Erika
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Forsgren, Anders
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    On dynamically identifying elementary flux modes for a poly-pathway model of metabolic reaction networksManuscript (preprint) (Other academic)
    Abstract [en]

    he aim with poly-pathway models is to model variations in the metabolic behavior of cells in response to changes in their external environment.By considering the elementary flux modes of a metabolic network, the network can be reduced to a set of macroscopic reactions. The macroscopic reactions connect external substrates to products, where each reaction is associated with a kinetic equation.Since enumerating all elementary flux modes is prohibitive for complex networks, these types of models are usually limited to simple networks. In this work we consider an algorithm for identifying elementary flux modes for a poly-pathway model. First we consider a dynamic identification of elementary flux modes and model parameters using column generation. However, due to non-linearity in one optimization problem involved in column generation, elementary flux mode identification can not be guaranteed with that column generation approach. In order to still be able to identify elementary flux modes, an approximation algorithm is derived and tested for the model identification. In a case study, the algorithm is shown to work well in practice and obtains a near-optimal solution compared to a method in which all elementary flux modes are enumerated beforehand.

  • 28.
    Oddsdóttir, Hildur Æsa
    et al.
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Hagrot, Erika
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Forsgren, Anders
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    On the Robustness of Elementary-Flux-Modes-based Metabolic Flux AnalysisManuscript (preprint) (Other academic)
    Abstract [en]

    Elementary flux modes (EFMs) are vectors defined from a metabolic reaction network, giving the connections between substrates and products. EFMs-based metabolic flux analysis (MFA) estimates the flux over each EFM from external flux measurements through least-squares data fitting. The measurements used in the data fitting are subject to errors. A robust optimization problem includes information on errors and gives a way to examine the sensitivity of the solution of the EFMs-based MFA to these errors. In general, formulating a robust optimization problem may make the problem significantly harder. We show that in the case of the EFMs-based MFA the robust problem can be stated as a convex quadratic programming problem. We have previously shown how the data fitting problem may be solved in a column-generation framework. In this paper, we show how column generation may be applied also to the robust problem. Furthermore, the option to indicate intervals on metabolites that are not measured is introduced in this column generation framework. The robustness of the data is evaluated in a case-study, which indicated that the solutions of our non-robust problems are in fact near-optimal also when robustness is considered, implying that the errors in measurement do not have a large impact on the optimal solution. Furthermore, we showed that the addition of intervals on unmeasured metabolites resulted in a change in the optimal solution.

  • 29.
    Oddsdóttir, Hildur Æsa
    et al.
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    Hagrot, Erika
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Chotteau, Véronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Forsgren, Anders
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Optimization and Systems Theory.
    On dynamically generating relevant elementary flux modes in a metabolic network using optimization2014In: Journal of Mathematical Biology, ISSN 0303-6812, E-ISSN 1432-1416Article in journal (Refereed)
    Abstract [en]

    Elementary flux modes (EFMs) are pathways through a metabolic reaction network that connect external substrates to products. Using EFMs, a metabolic network can be transformed into its macroscopic counterpart, in which the internal metabolites have been eliminated and only external metabolites remain. In EFMs-based metabolic flux analysis (MFA) experimentally determined external fluxes are used to estimate the flux of each EFM. It is in general prohibitive to enumerate all EFMs for complex networks, since the number of EFMs increases rapidly with network complexity. In this work we present an optimization-based method that dynamically generates a subset of EFMs and solves the EFMs-based MFA problem simultaneously. The obtained subset contains EFMs that contribute to the optimal solution of the EFMs-based MFA problem. The usefulness of our method was examined in a case-study using data from a Chinese hamster ovary cell culture and two networks of varied complexity. It was demonstrated that the EFMs-based MFA problem could be solved at a low computational cost, even for the more complex network. Additionally, only a fraction of the total number of EFMs was needed to compute the optimal solution.

  • 30. Ravichandran, R.
    et al.
    Åstrand, Carolina
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Patra, H. K.
    Turner, Anthony P. F.
    Chotteau, Véronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Phopase, J.
    Intelligent ECM mimetic injectable scaffolds based on functional collagen building blocks for tissue engineering and biomedical applications2017In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 7, no 34, 21068-21078 p.Article in journal (Refereed)
    Abstract [en]

    Hydrogels comprising natural extracellular matrix (ECM) components are very attractive as scaffolds for regenerative medicine applications due to their inherent biointeractive properties. Responsive materials that adapt to their surrounding environments and regulate transport of ions and bioactive molecules manifest significant advantages for biomedical applications. Although there are many exciting challenges, the opportunity to design, fabricate and engineer stimuli-responsive polymeric systems based on ECM components is particularly attractive for regenerative medicine. Here we describe a one-pot approach to fabricate in situ fast gellable intelligent ECM mimetic scaffolds, based on methacrylated collagen building blocks with mechanical properties that can be modulated in the kPa-MPa range and that are suitable for both soft and hard tissues. Physiochemical characterizations demonstrate their temperature and pH responsiveness, together with the structural and enzymatic resistance that make them suitable scaffolds for long-term use in regenerative medicine and biomedical applications. The multifunctionality of these hydrogels has been demonstrated as an in situ depot-forming delivery platform for the adjustable controlled release of proteins and small drug molecules under physiological conditions and as a structural support for adhesion, proliferation and metabolic activities of human cells. The results presented herein should be useful to the design and fabrication of tailor-made scaffolds with tunable properties that retain and exhibit sustained release of growth factors for promoting tissue regeneration.

  • 31.
    Zamani, Leila
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Aberg, Magnus
    Lindahl, Anna
    Mie, Axel
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Metabolic footprinting of CHO cell culture bioprocess data in fed-batch and perfusion mode using LC-MS data and multivariate analysisManuscript (preprint) (Other academic)
  • 32.
    Zhang, Ye
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Observation of Chinese Hamster Ovary Cells retained inside the non-woven fiber matrix of the CellTank bioreactor2015In: Data in Brief, ISSN 2352-3409, Vol. 5, 586-588 p.Article in journal (Refereed)
    Abstract [en]

    This data article shows how the recombinant Chinese Hamster Ovary (CHO) cells are located in the interstices of the matrix fibers of a CellTank bioreactor after completion of a perfusion culture, supporting the article entitled "Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor" by Zhang et al. [1]. It provides a visualization of the cell distribution in the non-woven fiber matrix in a deeper view.

  • 33.
    Zhang, Ye
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Stobbe, Per
    Silvander, Christian Orrego
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor2015In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 213, 28-41 p.Article in journal (Refereed)
    Abstract [en]

    Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200 pF/cm (where 1 pF/cm is equivalent to 1 x 106 viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37 C to 29 C was studied during 13 days at cell densities above 100 pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31 C was applied at cell density 100 pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period.

  • 34.
    Zhang, Ye
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Zhan, Caijuan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Girod, Pierre-Alain
    Martiné, Alexandra
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Optimization of the cell specific perfusion rate in high cell density perfusion processManuscript (preprint) (Other academic)
1 - 34 of 34
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