Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system

Nakamura, Tsuyoshi; Ômasa, Takeshi

doi:10.1016/j.jbiosc.2015.01.002

Cited by 35 publications

(27 citation statements)

References 42 publications

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“…In addition to the approach described here, other high‐throughput early clone characterization strategies have also been developed. ClonePix (Molecular Devices, Sunnyvale, CA) was able to automatically image, select, and pick mammalian cell colonies expanded in semi‐solid media, based on a number of parameters such as colony size and yield of a recombinant secreted product . However, the ClonePix readouts are not quantitative.…”

Section: Discussionmentioning

confidence: 99%

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Integration of high‐throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi‐subunit vaccine antigen

Li¹,

Zhang²,

Li³

et al. 2019

Biotechnology Progress

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Mammalian cell line generation typically includes stable pool generation, single cell cloning and several rounds of clone selection based on cell growth, productivity and product quality criteria. Individual clone expansion and phenotype‐based ranking is performed initially for hundreds or thousands of mini‐scale cultures, representing the major operational challenge during cell line development. Automated cell culture and analytics systems have been developed to enable high complexity clone selection workflows; while ensuring traceability, safety, and quality of cell lines intended for biopharmaceutical applications. Here we show that comprehensive and quantitative assessment of cell growth, productivity, and product quality attributes are feasible at the 200–1,200 cell colony stage, within 14 days of the single cell cloning in static 96‐well plate culture. The early cell line characterization performed prior to the clone expansion in suspension culture can be used for a single‐step, direct selection of high quality clones. Such clones were comparable, both in terms of productivity and critical quality attributes (CQAs), to the top‐ranked clones identified using an established iterative clone screening approach. Using a complex, multi‐subunit antigen as a model protein, we observed stable CQA profiles independently of the cell culture format during the clonal expansion as well as in the batch and fed‐batch processes. In conclusion, we propose an accelerated clone selection approach that can be readily incorporated into various cell line development workstreams, leading to significant reduction of the project timelines and resource requirements.

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Section: Discussionmentioning

confidence: 99%

“…ClonePix (Molecular Devices, Sunnyvale, CA) was able to automatically image, select, and pick mammalian cell colonies expanded in semi-solid media, based on a number of parameters such as colony size and yield of a recombinant secreted product [22][23][24]. However, the ClonePix readouts are not quantitative.…”

mentioning

confidence: 99%

Integration of high‐throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi‐subunit vaccine antigen

Li¹,

Zhang²,

Li³

et al. 2019

Biotechnology Progress

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show abstract

“…27 New techniques for the generation of stable cell lines have reduced the timeline, but these generally involve the use of costly equipment, such as flow cytometers capable of sorting or automated colony picking (i.e., CloniPix FL). [39][40][41][42] We produced a stable cell pool in less than 5 weeks and stable clones in 10 weeks using the methods described here. We extended the expression and improved antibody production using puromycin to kill low-producer cells and to select for stable pools and clones of highproducer cells (Figure 2A).…”

Section: Generation Of Hek293 Cells With Stable Expression Of Mab13c6mentioning

confidence: 99%

Rapid and cost-effective development of stable clones for the production of anti-Ebola monoclonal antibodies in HEK293T cells

Gonzalez‐González

Palestino-Díaz

López-Pacheco

et al. 2020

Preprint

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The Ebola virus (EBOV) disease has caused serious and recurrent epidemics in recent years, resulting in a fatality rate of nearly 50%. The most effective experimental therapy against the EBOV is the use of monoclonal antibodies (mAbs). In this work, we describe the development of HEK293T cells engineered for the transient and stable expression of mAb13C6, a neutralizing anti-EBOV monoclonal antibody. We transfected the HEK293T cells with a tricistronic vector to produce the heavy and the light chain of the antibody 13C6 and intracellular Green Fluorescent Protein (GFP) using Lipofectamine 3000. We then selected the transfected cells using puromycin pressure, dilution cloning, and cloning disks. This integrated strategy generated mAb-producing cells in 7 days with a transient expression of ~1 mg/L. Stable pools were produced after 4 weeks, with expression levels of ~0.8 mg/L. Stable clones with expression levels of ~1.8 mg/L were obtained within 10 weeks. The produced antibodies exhibited the expected functionality; they recognized the GP glycoprotein of the Ebola virus in both ELISA assays and cell binding experiments using HEK293T cells engineered to express the EBOV GP at their membrane surface. By the combined use of GFP and the set of selection techniques here described, we drastically reduced the time from transfection to stable clone generation without resorting to costly equipment. In outbreaks or emergencies, this platform can significantly shorten the development of new biopharmaceuticals and vaccines.

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“…Single cells are labelled through binding of antibody to the membrane associated recombinant protein, and fluorescence-activated cell sorting (FACS) is used to separate small percentage of clones with high secretion rates 53 . Another method involves suspending single cells in a semi solid medium to enable accumulation of secreted protein in the vicinity of the clone followed by quantification of that protein using a fluorescently labelled antibody 54 . High producing clones can be identified based on fluorescence detection and expanded.…”

Section: Strategies For High and Medium Throughput Clone Screeningmentioning

confidence: 99%

Cell Culture Processes for Biopharmaceutical Manufacturing

Gadgil

2017

Current Science

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Recombinant proteins manufactured using animal cell culture processes comprise a significant fraction of biopharmaceuticals. With the expiry of patents on this class of therapeutics, there is also a significant interest in manufacture of biosimilar versions of such therapeutics. This article provides a birds-eye view of upstream process development for animal cell culture processes, with a focus on advances pertinent to the development of processes for biosimilars.

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Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system

Cited by 35 publications

References 42 publications

Integration of high‐throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi‐subunit vaccine antigen

Integration of high‐throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi‐subunit vaccine antigen

Rapid and cost-effective development of stable clones for the production of anti-Ebola monoclonal antibodies in HEK293T cells

Cell Culture Processes for Biopharmaceutical Manufacturing

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