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

Dharshanan, Suba; Chong, Heilly; Cheah, Swee Hung; Zamrod, Zulkeflie

doi:10.1007/s10616-013-9615-x

Cited by 13 publications

(7 citation statements)

References 12 publications

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“…In contrast, a decrease in the cellspecific productivity was observed in two of the non-UCOE cell lines. These results are consistent with previous studies, where inclusion of UCOE in the vector construct resulted in higher levels of recombinant protein production [19,21,27,28,30] and improved the transgene expression in hematopoietic stem cells [25,26]. Otte et al compared the effect of different DNA elements, including UCOEs, S/MARs, cHS4 insulator and anti-repressor elements, on protein expression levels [37].…”

Section: Discussionsupporting

confidence: 90%

“…Anti-silencing activity was associated with strongly reduced DNA methylation of promoter, which strongly favours its application in PSC-based cell and gene therapy. In CHO and BHK21 cells, UCOE provided a higher level of transgene expression following stable transfection [19,21,[27][28][29] and maintained the stability of protein expression over 100 generations [30]. In this study, a UCOE vector was used in combination with MTX amplification to improve the frequency of positive clones and to achieve a high volumetric recombinant protein production.…”

Section: Introductionmentioning

confidence: 99%

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Assessment of UCOE on Recombinant EPO Production and Expression Stability in Amplified Chinese Hamster Ovary Cells

Betts

Dickson

2015

Mol Biotechnol

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CHO cells are the most frequently used host for commercial production of therapeutic proteins, and DHFR-mediated gene amplification is extensively applied to generate cell lines with increased protein production. However, decreased protein productivity is observed unpredictably during the time required for scale-up with consequences for yield, time, finance and regulatory approval. In this study, we have examined the interaction between Ubiquitous Chromatin Opening Elements (UCOE) and DHFR-linked amplification in relation to cell expression stability. In summary, the inclusion of UCOE elements generated cells that (1) achieved higher cell densities and exhibited increased production of recombinant mRNA per cell and protein yield, (2) allowed isolation of greater numbers of high-producing clones, (3) resulted in greater mRNA recovery per recombinant gene copy, (4) retained stable mRNA and protein expression after amplification provided Methotrexate (MTX) was present (but not in the absence of MTX when instability was observed) and (5) conferred copy number-dependent expression to linked transgene, suggesting that they are resistant to positional gene-silencing effects. It was concluded that the inclusion of UCOEs within expression constructs offers significant advantages for certainty of cell line generation (and the number of recovered clones for more detailed characterisation/optimisation) and that UCOEs are compatible with DHFR amplification protocols. The data suggested that enhanced cell line recovery by transcriptional enhancement of selection markers, such as DHFR, could be achieved.

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Section: Discussionsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Assessment of UCOE on Recombinant EPO Production and Expression Stability in Amplified Chinese Hamster Ovary Cells

Betts

Dickson

2015

Mol Biotechnol

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“…Studies so far have shown that in CHO and BHK21 cells, UCOE provided a higher level of transgene expression following stable transfection and maintained the stability of protein expression over 100 generations . Often recombinant protein production in CHO cells is enhanced by amplifying the gene of interest via methrotrexate (MTX) amplification.…”

Section: Introductionmentioning

confidence: 99%

Evaluating the interaction between UCOE and DHFR‐linked amplification and stability of recombinant protein expression

Betts

Croxford

Dickson

2015

Biotechnology Progress

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Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry. In the creation of mammalian cell lines plasmid DNA carrying the gene-of-interest integrates randomly into the host cell genome, which results in variable levels of gene expression between cell lines due to gene silencing mechanisms. In addition, cell lines often show unstable protein production during long-term culture. This means that a large number of clones need to be screened in order to isolate stable, high producing cell lines making mammalian cell line development a long and laborious process. In this study an expression platform incorporating a Ubiquitous Chromatin Opening Element (UCOE; which are proposed to maintain chromatin in an open state) has been utilised for the expression of eGFP in CHO cells. Cell lines containing a UCOE vector, showed a significantly higher and more consistent eGFP expression than the non-UCOE cell lines without DHFR amplification. To further improve recombinant protein production cell lines were amplified with methotrexate (MTX). UCOE cell lines showed improved growth in MTX therefore amplification to 250 nM MTX was achieved following a one-step amplification procedure. However, non-UCOE cell lines showed higher levels of eGFP production following MTX amplification. In addition, UCOE cell lines did not improve stability during long-term culture in the absence of selective pressure. Stable eGFP production was achieved for all cell lines when MTX is present. Finally, UCOE cell lines displayed more consistent response to external stimuli than non-UCOE cell lines, suggesting that UCOE cell lines are less prone to clonal variability.

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“…With this innovative system (termed IR/MAR-DHFR) CHO cell lines (both DUXB11 and DG44 backgrounds) can be created that produce more recombinant protein and show a degree of enhanced stability over long term culture in comparison with the standard DHFR -/MTX system on its own. Finally, UCOEs have also been utilised within vector designs to enhance the stability of stable CHO clones and also to boost the productivity from stable transfectants pools (Dharshanan et al 2013;Ye et al 2010;Kwaks and Otte 2006). UCOEs are genetic elements isolated from the upstream regions of housekeeper genes and include unmethylated CpG islands and other promoter-associated elements.…”

Section: Strategies To Combat Cell Line Instabilitymentioning

confidence: 99%

Building a Cell Culture Process with Stable Foundations: Searching for Certainty in an Uncertain World

O'Callaghan¹,

Racher

2014

Cell Engineering

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Considerable effort is expended during the mammalian cell line construction process to find a stably-transfected clone capable of supporting the largescale manufacture of a recombinant therapeutic protein. Such a clone must synthesise a sufficient volumetric concentration of the product with the correct biochemical characteristics (glycosylation, structural integrity, etc.). Furthermore, this performance must be maintained over the extended time period required to support a manufacturing campaign in 20,000 L bioreactors. However, a significant proportion of recombinant clonal cell lines show production instability over longterm sub-culture, where volumetric product yield and/or product quality are not maintained. This instability can potentially extend product development timelines and can affect the ability of a manufacturing process to meet market demand. In the worse-case scenario it can also jeopardise patient safety if product quality is impaired. In order to prevent this, industrial cell line construction processes include long-term stability studies where several candidate lead clones are serially sub-cultured and monitored for signs of instability before selecting the final production cell line. The roots of production instability are varied, but the epigenetic silencing of transgenes at sites of host cell chromosome integration, and the direct mutation or loss of transgenes are prominent molecular causes. Considerable research has been conducted by both industry and academic groups into the molecular mechanisms underpinning instability, with an emphasis on uncovering early predictive markers of incipient instability as well as preventing its occurrence. In this article we present a detailed overview of the industrial experience of production instability and its impact on the manufacturing of recombinant therapeutics. We also discuss our current understanding of the molecular causes of cell line instability and how this has been used to mitigate the impact of this phenomenon through novel vector redesigns and cell line screening.

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

Cited by 13 publications

References 12 publications

Assessment of UCOE on Recombinant EPO Production and Expression Stability in Amplified Chinese Hamster Ovary Cells

Assessment of UCOE on Recombinant EPO Production and Expression Stability in Amplified Chinese Hamster Ovary Cells

Evaluating the interaction between UCOE and DHFR‐linked amplification and stability of recombinant protein expression

Building a Cell Culture Process with Stable Foundations: Searching for Certainty in an Uncertain World

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