Selection methods for high-producing mammalian cell lines

Browne, Susan M.; Al‐Rubeai, Mohamed

doi:10.1016/j.tibtech.2007.07.002

Cited by 217 publications

(103 citation statements)

References 38 publications

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“…Mammalian cells, despite having lower yields, continue to be the system of choice because the other systems cannot adequately reproduce the post-translational modifications and glycosylation patterns that may be important for the tertiary structure of proteins (Browne and Al-Rubeai 2007). This is a vital factor because the lack of fidelity can lead to altered protein stability, lowered affinity to antigen, more rapid clearance rate and increased immunogenicity.…”

Section: Introductionmentioning

confidence: 99%

“…Thus, Chinese hamster ovary (CHO) and NS0 cell lines have been most commonly used for the production of therapeutic antibodies (Zhu 2011). Nevertheless, the production of therapeutic proteins in mammalian cells is an expensive process with long development time (Browne and Al-Rubeai 2007). Hence, in order to increase culture yields and stability and at the same time decrease the duration for the development and production of humanized mAbs, it is crucial to optimize the in vitro mammalian cell production system.…”

Section: Introductionmentioning

confidence: 99%

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Stable expression of H1C2 monoclonal antibody in NS0 and CHO cells using pFUSE and UCOE expression system

Dharshanan

Chong²,

Cheah

et al. 2013

Cytotechnology

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From our recent publications, it was found that the deimmunization method (Dharshanan et al. (2012) Sci Res Essays 7:2288–2299) should be applied for the development of humanized anti-C2 monoclonal antibody (H1C2 mAb). However, the overlapping-PCR mutagenesis procedure used to insert the variable regions into cloning vectors was laborious and time-consuming. Additionally, the expression of H1C2 mAb in NS0 cells was low in static culture vessels. Therefore H1C2 mAb was redeveloped by deimmunization method with the following modifications in order to optimize the production of H1C2 mAb. First, instead of the overlapping-PCR mutagenesis procedure, synthetic DNA coding the variable regions were used to express the mAb. Second, two expression vectors, pFUSE and UCOE, were used to express H1C2 mAb in NS0 cells and CHO cells in order to investigate the combination that gave the highest number of high producing stable clones. This will provide the highest chance of finding clones with the requisite high productivity and stability required for manufacturing. We found that transfection of UCOE in CHO cells generated the highest number of high producing stable clones. To our knowledge, this is the first time that H1C2 mAb has been expressed in CHO cells.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Stable expression of H1C2 monoclonal antibody in NS0 and CHO cells using pFUSE and UCOE expression system

Dharshanan

Chong²,

Cheah

et al. 2013

Cytotechnology

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“…Fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS) have been commonly used to analyze large populations of suspended cells with biochemical characteristics of cell surface markers12. Microfabrication technology3, microfluidic devices45, and optical trapping6 allow to manipulate small numbers of floating cells in suspension and to attach cells onto micropatterned two-dimensional (2D) surfaces.…”

mentioning

confidence: 99%

Optical cell separation from three-dimensional environment in photodegradable hydrogels for pure culture techniques

Tamura

Yanagawa

Sugiura

et al. 2014

Sci Rep

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Cell sorting is an essential and efficient experimental tool for the isolation and characterization of target cells. A three-dimensional environment is crucial in determining cell behavior and cell fate in biological analysis. Herein, we have applied photodegradable hydrogels to optical cell separation from a 3D environment using a computer-controlled light irradiation system. The hydrogel is composed of photocleavable tetra-arm polyethylene glycol and gelatin, which optimized cytocompatibility to adjust a composition of crosslinker and gelatin. Local light irradiation could degrade the hydrogel corresponding to the micropattern image designed on a laptop; minimum resolution of photodegradation was estimated at 20 µm. Light irradiation separated an encapsulated fluorescent microbead without any contamination of neighbor beads, even at multiple targets. Upon selective separation of target cells in the hydrogels, the separated cells have grown on another dish, resulting in pure culture. Cell encapsulation, light irradiation and degradation products exhibited negligible cytotoxicity in overall process.

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“…It has been reported that the specific productivity can be increased significantly by the gene amplification using MTX [10]. Furthermore, many advanced methods have been recently developed for the efficient selection of highproducing clones among the thousands of clones [11]. For example, there is a new technology where cells expressing a target protein fused to a fluorescent protein can be selectively collected using flow cytometry.…”

Section: Selection Of High-productive Clonesmentioning

confidence: 99%

Cellular engineering for the high-level production of recombinant proteins in mammalian cell systems

Park

2010

Korean J. Chem. Eng.

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The market for protein-drugs has steadily increased due their increased use as alternatives to traditional small molecule drugs. While some therapeutic proteins have been produced in microbial systems, mammalian cell systems such as Chinese hamster ovary (CHO) cells are widely used as the host cell system. To increase the efficiency of producing therapeutic proteins, many researchers have attempted to solve the critical problems that occur in mammalian cell systems. As a result, several serum-free media and advanced culture methods have been developed, and protein productivity has increased considerably through the development of efficient selection methods. However, the prevalence of apoptosis during mammalian cell culture still remains a significant problem. Based on the understanding of apoptotic mechanisms and related proteins, anti-apoptotic engineering has steadily progressed. In this study, we review the strategies that have been developed for high-level production of recombinant proteins in the CHO cell system via a selection of clones, target-gene amplification, optimization of culture systems and an inhibition of apoptosis through genetic modification.

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Selection methods for high-producing mammalian cell lines

Cited by 217 publications

References 38 publications

Stable expression of H1C2 monoclonal antibody in NS0 and CHO cells using pFUSE and UCOE expression system

Stable expression of H1C2 monoclonal antibody in NS0 and CHO cells using pFUSE and UCOE expression system

Optical cell separation from three-dimensional environment in photodegradable hydrogels for pure culture techniques

Cellular engineering for the high-level production of recombinant proteins in mammalian cell systems

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