Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems

Müller, Natalie; Derouazi, Madiha; Tilborgh, Frédéric Van; Wulhfard, Sarah; Hacker, David L.; Jordan, Martin; Wurm, Florian M.

doi:10.1007/s10529-006-9298-x

Cited by 72 publications

(54 citation statements)

References 23 publications

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“…PEI is a cationic polymer that condenses DNA into small particles or interacts with medium components (Muller et al 2007). If PEI has higher affinity for these components than for the DNA the transfection efficiency decreases (Godbey et al 1999).…”

Section: Discussionmentioning

confidence: 99%

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

et al. 2009

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The generation of transgenic cell lines is acquired by facilitating the uptake and integration of DNA. Unfortunately, most of the systems generating stable expression systems are cost and time-consuming and transient expression is optimized to generate milligram amounts of the recombinant protein.Therefore we improved and compared two transfection systems, one based on cationic liposomes consisting of DOTAP/DOPE and the second one on polyethylenimine (PEI). Both systems have been used as chemically defined transfection systems in combination with serum-free cultivated host cell line. At first we had determined the toxicity and ideal ratio of DNA to PEI followed by determination of the optimal transfection conditions in order to achieve maximum transfection efficiency. We then directly compared DOTAP/DOPE and PEI in transient transfection experiments using enhanced green fluorescence protein (EGFP) and a human monoclonal antibody, mAb 2F5, as a model protein. The results which were achieved in case of EGFP were more than 15% transfectants at a viability of 85%. Despite the fact that expression of the mAb was found negligible we used both techniques to generate stable mAb 2F5 expressing cell lines that underwent several cycles of screening and amplification with methotrexate, and resulted in cell lines with similar volumetric production titers. These experiments serve to demonstrate the potential of stable cell lines even in case where the transient systems did not show satisfying results.

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Section: Discussionmentioning

confidence: 99%

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

et al. 2009

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“…The immunocytokines F8-hp35/hp40-F8, hIL12-F8-F8, hIL12-F8 diabody, mIL12-F8-F8, and mIL12-KSF-KSF were expressed using transient gene expression as described before (39,40). For 1 mL of production 1 Â 10 6 CHO-S cells in suspension were centrifuged and resuspended in 0.5 mL ProCHO4 (Lonza).…”

Section: Cloning Of Fusion Proteinsmentioning

confidence: 99%

“…At 4 hour posttransfection, the transfected culture was diluted with 0.5 mL of Power-CHO-2CD and then incubated at 31 C in a shaker incubator for 6 days. The procedure was scaled up to reach the desired production volume (40).…”

Section: Cloning Of Fusion Proteinsmentioning

confidence: 99%

The Antibody-Based Delivery of Interleukin-12 to the Tumor Neovasculature Eradicates Murine Models of Cancer in Combination with Paclitaxel

Pasche

Wulhfard

Pretto

et al. 2012

Clinical Cancer Research

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104

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Purpose: Interleukin-12 (IL12) is a potent proinflammatory cytokine with antitumor activity. Its heterodimeric nature makes it compatible with a large variety of different immunocytokine formats. Here we report the design, production, and characterization of a novel immunocytokine, based on the fusion of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin, a marker of tumor neovasculature) with IL12 (termed IL12-F8-F8).Experimental Design: We developed a novel immunocytokine based on the sequential fusion of interleukin-12 as a single polypeptide with two F8 antibodies in single-chain Fv (scFv) format. The fusion protein was characterized in vitro, and its targeting performance was assessed in vivo. The immunocytokine antitumor activity was studied as monotherapy as well as in combination therapies in three different murine tumor models. Moreover, depletion experiments and tumor analysis revealed a dominant role of natural killer cells for the mechanism of action.Results: IL12-F8-F8 can be produced in mammalian cells, yielding a product of good pharmaceutical quality, capable of selective localization on the tumor neovasculature in vivo, as judged by quantitative biodistribution analysis with radioiodinated protein preparations. The protein potently inhibited tumor growth in three different immunocompetent syngeneic models of cancer. The treatment was generally well tolerated. Moreover, the IL12-F8-F8 fusion protein could be produced both with murine IL12 (mIL12) and with human IL12 (hIL12).Conclusions: The potent antitumor activity of mIL12-F8-F8, studied alone or in combination with paclitaxel in different tumor models, paves the way to the clinical development of the fully human immunocytokine. Clin Cancer Res; 18(15); 4092-103. Ó2012 AACR.

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“…These reasons kindled the interest in the use of large scale orbital shaken devices up to thousand liters. [7][8][9] Thus, a large number of researches have been performed on the gas exchange, [10][11][12][13] mixing, [14][15][16][17] and volumetric power consumption 18 of the bioreactors. In some cases, correlations have been proposed in order to quantify the variation of those quantities during scale-up of the culture size.…”

Section: Introductionmentioning

confidence: 99%

Surface wave dynamics in orbital shaken cylindrical containers

et al. 2014

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Be it to aerate a glass of wine before tasting, to accelerate a chemical reaction, or to cultivate cells in suspension, the "swirling" (or orbital shaking) of a container ensures good mixing and gas exchange in an efficient and simple way. Despite being used in a large range of applications this intuitive motion is far from being understood and presents a richness of patterns and behaviors which has not yet been reported. The present research charts the evolution of the waves with the operating parameters identifying a large variety of patterns, ranging from single and multiple crested waves to breaking waves. Free surface and velocity fields measurements are compared to a potential sloshing model, highlighting the existence of various flow regimes. Our research assesses the importance of the modal response of the shaken liquids, laying the foundations for a rigorous mixing optimization of the orbital agitation in its applications. C 2014 AIP Publishing LLC. [http://dx

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Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems

Cited by 72 publications

References 23 publications

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

The Antibody-Based Delivery of Interleukin-12 to the Tumor Neovasculature Eradicates Murine Models of Cancer in Combination with Paclitaxel

Surface wave dynamics in orbital shaken cylindrical containers

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