Efficient selection of high-producing subclones during gene amplification of recombinant Chinese hamster ovary cells by flow cytometry and cell sorting

Borth, Nicole; Zeyda, Maximilian; Katinger, Hermann

doi:10.1002/1097-0290(2000)71:4<266::aid-bit1016>3.0.co;2-2

Cited by 81 publications

(51 citation statements)

References 32 publications

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“…Previous flow cytometric studies of hybridoma cells aimed to detect antibodies on the cell surface using only fluorescein-labeled anti-mouse IgG (Marder et al, 1990;Sen et al, 1990;McKinney et al, 1991;Kromenaker and Srienc, 1994;Cherlet et al, 1995;Borth et al, 2000). The lack of information on the specificity of the hybridoma cells, however, is the major disadvantage of this method and therefore it has not been used on a routine basis, yet.…”

Section: Discussionmentioning

confidence: 99%

“…In order to identify these cells thousands of clones need to be analyzed, which is impossible by traditional methods (Browne and Al-Rubeai, 2007). A high-throughput technique such as flow cytometry and cell sorting would be suitable in the selection of these rare cells (Borth et al, 2000). Our approach employs the technique of antigen-specific labeling of cells, which allows fluorescence activated cell sorting and thereby eliminate the abovementioned problems.…”

Section: Discussionmentioning

confidence: 99%

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Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA

Kuhne

Dippong

Flemig

et al. 2014

Journal of Immunological Methods

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA

Kuhne

Dippong

Flemig

et al. 2014

Journal of Immunological Methods

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“…Flow cytometry is a versatile and valuable tool in research on mammalian cell cultures (Al-Rubeai et al 1991b;Borth et al 2000;Carroll and Al-Rubeai 2004;Lee and Lee 2012). The execution of flow cytometry experiments traditionally involves several manual sample preparation steps, depending on the protocol.…”

Section: Introductionmentioning

confidence: 99%

Online flow cytometry for monitoring apoptosis in mammalian cell cultures as an application for process analytical technology

2014

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Apoptosis is the main driver of cell death in bioreactor suspension cell cultures during the production of biopharmaceuticals from animal cell lines. It is known that apoptosis also has an effect on the quality and quantity of the expressed recombinant protein. This has raised the importance of studying apoptosis for implementing culture optimization strategies. The work here describes a novel approach to obtain near real time data on proportion of viable, early apoptotic, late apoptotic and necrotic cell populations in a suspension CHO culture using automated sample preparation in conjunction with flow cytometry. The resultant online flow cytometry data can track the progression of apoptotic events in culture, aligning with analogous manual methodologies and giving similar results. The obtained near-real time apoptosis data are a significant improvement in monitoring capabilities and can lead to improved control strategies and research data on complex biological systems in bioreactor cultures in both academic and industrial settings focused on process analytical technology applications.

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“…However, the process of randomly picking a large number of clones (e.g. by limited dilution and individually assaying them by ELISA) is boring, time-wasting, and reportedly not always very effective (Borth et al 2001;Zeyda et al 1999). Moreover, this method can not take factors like differences in either cell density or medium volume between wells into consideration.…”

Section: Introductionmentioning

confidence: 99%

Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1

et al. 2014

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Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods.

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Efficient selection of high-producing subclones during gene amplification of recombinant Chinese hamster ovary cells by flow cytometry and cell sorting

Cited by 81 publications

References 32 publications

Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA

Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA

Online flow cytometry for monitoring apoptosis in mammalian cell cultures as an application for process analytical technology

Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1

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