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  • 1.
    Al-Khalili, Lubna
    et al.
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Gillner, Karin
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Zhang, Ye
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Åstrand, Carolina
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Shokri, Atefeh
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Hughes-Brittain, Nanaaya
    McKean, Robert
    Robb, Brendan
    Chotteau, Véronique
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Characterization of Human CD133+Cells in Biocompatible Poly(l-lactic acid) Electrospun Nano-Fiber Scaffolds2016Ingår i: Journal of Biomaterials and Tissue Engineering, ISSN 2157-9083, E-ISSN 2157-9091, Vol. 6, nr 12, s. 959-966Artikel i tidskrift (Refereegranskat)
    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.
    Chotteau, Veronique
    et al.
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Zhang, Ye
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Clincke, Marie-Francoise
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Very High Cell Density in Perfusion of CHO Cells by ATF, TFF, Wave Bioreactor, and/or CellTank Technologies: Impact of Cell Density and Applications2014Ingår i: Continuous Processing in Pharmaceutical Manufacturing / [ed] Ganapathy Subramanian, Germany Weinheim: Wiley-VCH Verlagsgesellschaft, 2014, s. 339-356Kapitel i bok, del av antologi (Övrigt vetenskapligt)
  • 3.
    Clincke, Marie-Francoise
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Mölleryd, Carin
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Samani, Puneeth K
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Zhang, Ye
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Lindskog, Eva
    GE Healthcare, Uppsala, Sweden.
    Walsh, Kieron
    GE Healthcare, Westborough, MA, USA.
    Chotteau, Veronique
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Perfusion of an IgG producing CHO cell line at very high cell density by ATF or by TFF in WAVE Bioreactor™2011Konferensbidrag (Övrigt vetenskapligt)
    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.

  • 4.
    Clincke, Marie-Francoise
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Mölleryd, Carin
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Samani, Puneeth K.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Zhang, Ye
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Lindskog, Eva
    GE Healthcare, Uppsala, Sweden.
    Walsh, Kieron
    GE Healthcare, Westborough, MA, USA.
    Chotteau, Veronique
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Perfusion of an IgG producing CHO cell line at very high cell density by ATF or by TFF in WAVE Bioreactor™2011Konferensbidrag (Övrigt vetenskapligt)
    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.

  • 5.
    Clincke, Marie-Francoise
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Mölleryd, Carin
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Zhang, Ye
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Lindskog, Eva
    GE Healthcare, Uppsala, Sweden.
    Walsh, Kieron
    GE Healthcare, Westborough, MA, USA.
    Chotteau, Veronique
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Study of a recombinant CHO cell line producing a monoclonal antibody by ATF or TFF external filter perfusion in a WAVE Bioreactor™2011Ingår i: BMC Proceedings, 2011, Volume 5, Supplement 8, P105, BioMed Central, 2011, s. 105-Konferensbidrag (Refereegranskat)
    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. 

  • 6.
    Clincke, Marie-Francoise
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Mölleryd, Carin
    KTH, Skolan för bioteknologi (BIO).
    Zhang, Ye
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik (stängd 20130101).
    Lindskog, Eva
    Walsh, Kieron
    Chotteau, Veronique
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik (stängd 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 process2013Ingår i: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 29, nr 3, s. 754-767Artikel i tidskrift (Refereegranskat)
    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.

  • 7.
    Schwarz, Hubert
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Zhang, Ye
    KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Zhan, Caijuan
    KTH, Skolan för teknikvetenskap (SCI). KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Malm, Magdalena
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Field, Raymond
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Turner, Richard
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Sellick, Christopher
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Varley, Paul
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Chotteau, Véronique
    KTH, Skolan för teknikvetenskap (SCI).
    Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant ErythropoietinManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Process intensification in mammalian cell culture-based recombinant protein production has been achieved by high cell density perfusion exceeding 108 cells/mL in the recent years. As the majority of therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cells, intensified perfusion processes have been mainly developed for this type of host cell line. However, the use of CHO cells can result in non-human posttranslational modifications of the protein of interest, which may be disadvantageous compared with human cell lines.

    In this study, we developed a high cell density perfusion process of Human Embryonic Kidney (HEK293) cells producing recombinant human Erythropoietin (rhEPO). Firstly, a small-scale perfusion system from commercial bench-top screening bioreactors was developed for <250 mL working volume. Then, after the first trial runs with CHO cells, the system was modified for HEK293 cells (more sensitive than CHO cells) to achieve a higher oxygen transfer under mild aeration and agitation conditions. Steady states for medium (20 x 106 cells/mL) and high cell densities (80 x 106 cells/mL), normal process temperature (37 °C) and mild hypothermia (33 °C) as well as different cell specific perfusion rates (CSPR) from 10 to 60 pL/cell/day were applied to study the performance of the culture. The volumetric productivity was maximized for the high cell density steady state but decreased when an extremely low CSPR of 10 pL/cell/day was applied. The shift from high to low CSPR strongly reduced the nutrient uptake rates. The results from our study show that human cell lines, such as HEK293 can be used for intensified perfusion processes. 

  • 8.
    Zamani, Leila
    et al.
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Zhang, Ye
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Aberg, Magnus
    Lindahl, Anna
    Mie, Axel
    Chotteau, Veronique
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Metabolic footprinting of CHO cell culture bioprocess data in fed-batch and perfusion mode using LC-MS data and multivariate analysisManuskript (preprint) (Övrigt vetenskapligt)
  • 9.
    Zhang, Ye
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    High cell density perfusion process development for antibody producing Chinese Hamster Ovary cells2017Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Perfusion operation mode is currently under fast expansion in mammalian cell based manufacturing of biopharmaceuticals, not only for labile drug protein but also for stable proteins such as monoclonal antibodies (mAbs). Perfusion mode can advantageously offer a stable cell environment, long-term production with high productivity and consistent product quality. Intensified high cell density culture (HCDC) is certainly one of the most attractive features of a perfusion process due to the high volumetric productivity in a small footprint that it can provide. Advancements in single-use technology have alleviated the intrinsic complexity of perfusion processes while the maturing in cell retention devices has improved process robustness. The knowledge for perfusion process has been gradually built and the “continuous” concept is getting more and more acceptance in the field.

    This thesis presents the development of robust perfusion process at very high cell densities in various culture systems. Four HCDC perfusion systems were developed with industrial collaborators with three different mAb producing Chinese Hamster Ovary (CHO) cell lines: 1-2) WAVE Bioreactor™ Cellbag prototype equipped with cell separation by hollow fiber filter utilizing Alternating Tangential Flow (ATF) and Tangential Flow Filtration (TFF) techniques; 3) Fiber matrix based CellTank™ prototype; 4) Glass stirred tank bioreactor equipped with ATF. In all the systems, extremely high viable cell densities above 130 million viable cells per milliliter (MVC/mL) up to 214 MVC/mL were achieved. Steady states were maintained and studied at 20-30 MVC/mL and 100-130 MVC/mL for process development. Perfusion rate selection based on cell specific perfusion rate (CSPR) was systematically investigated and exometabolome study was performed to explore the metabolic footprint of HCDC perfusion process.

  • 10.
    Zhang, Ye
    et al.
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Chotteau, Veronique
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Observation of Chinese Hamster Ovary Cells retained inside the non-woven fiber matrix of the CellTank bioreactor2015Ingår i: Data in Brief, ISSN 2352-3409, Vol. 5, s. 586-588Artikel i tidskrift (Refereegranskat)
    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.

  • 11.
    Zhang, Ye
    et al.
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Stobbe, Per
    Silvander, Christian Orrego
    Chotteau, Veronique
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor2015Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 213, s. 28-41Artikel i tidskrift (Refereegranskat)
    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.

  • 12.
    Zhang, Ye
    et al.
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Zhan, Caijuan
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Girod, Pierre-Alain
    Martiné, Alexandra
    Chotteau, Veronique
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Optimization of the cell specific perfusion rate in high cell density perfusion processManuskript (preprint) (Övrigt vetenskapligt)
1 - 12 av 12
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  • en-GB
  • en-US
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  • rtf