miRNA profiling of high, low and non-producing CHO cells during biphasic fed-batch cultivation reveals process relevant targets for host cell engineering

Stiefel, Fabian; Fischer, Simon; Sczyrba, Alexander; Otte, Kerstin; Hesse, Friedemann

doi:10.1016/j.jbiotec.2016.03.028

Cited by 23 publications

(17 citation statements)

References 66 publications

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“…Previous miRNA profiling studies suggested that miR‐557 is not expressed in CHO cells (Diendorfer et al, ; Hackl et al, ; Hammond et al, ; Hernandez Bort et al, ; Lin et al, ; Stiefel et al, ), which we could confirm via qRT‐PCR based quantification of mature miR‐557 in our stable negative control miRNA expressing CHO host cells. According to the latest release of the miRBase repository (Kozomara and Griffiths‐Jones, ), miR‐557 has yet only found to be expressed in four species comprising human, chimpanzee, rhesus macaque, and orangutan.…”

Section: Discussionsupporting

confidence: 78%

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

Fischer

Marquart

Pieper

et al. 2017

Biotech & Bioengineering

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In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult‐to‐express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi‐specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high‐yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro‐productive miRNA: miR‐557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR‐557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR‐557 increased the probability to identify high‐producing cell clones. Furthermore, production cell lines derived from miR‐557 expressing host cells exhibited significantly increased final product yields in fed‐batch cultivation processes without compromising product quality. Strikingly, cells co‐expressing miR‐557 and a DTE antibody achieved a twofold increase in product titer compared to clones co‐expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495–1510. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

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Section: Discussionsupporting

confidence: 78%

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

Fischer

Marquart

Pieper

et al. 2017

Biotech & Bioengineering

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“…Our analyses of temporal miR‐143 expression demonstrated that expression levels of both mature miR‐143‐5p and −3p strands increased over time. Very recently, miR‐143‐3p was identified to be substantially up‐regulated in mAb‐producing CHO cells cultivated under mild hypothermic culture conditions in controlled bioreactors . Mild hypothermia has been frequently demonstrated to increase recombinant protein production in CHO cells .…”

Section: Discussionmentioning

confidence: 99%

“…Very recently, miR-143-3p was identified to be substantially up-regulated in mAb-producing CHO cells cultivated under mild hypothermic culture conditions in controlled bioreactors. 57 Mild hypothermia has been frequently demonstrated to increase recombinant protein production in CHO cells. [58][59][60] Taken together, all these data indicate that overexpression of miR-143 may modulate the cellular transcriptome toward a hyperproductive phenotype in CHO cells which is both observed during the stationary growth phase of a bioprocess but also as a result of low temperature cultivation.…”

Section: Mir-143 Represents a Novel Pro-productive Mirna In Cho Cellsmentioning

confidence: 99%

miR‐143 targets MAPK7 in CHO cells and induces a hyperproductive phenotype to enhance production of difficult‐to‐express proteins

Schoellhorn

Fischer

Wagner

et al. 2017

Biotechnology Progress

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In recent years, the number of complex but clinically effective biologicals such as multi-specific antibody formats and fusion proteins has increased dramatically. However, compared to classical monoclonal antibodies (mAbs), these rather artificially designed therapeutic proteins have never undergone millions of years of evolution and thus often turn out to be difficult-to-express using mammalian expression systems such as Chinese hamster ovary (CHO) cells. To provide access to these sophisticated but effective drugs, host cell engineering of CHO production cell lines represents a promising approach to overcome low production yields. MicroRNAs (miRNAs) have recently gained much attention as next-generation cell engineering tools. However, only very little is known about the capability of miRNAs to specifically increase production of difficult-to-express proteins. In a previous study we identified miR-143 amongst others to improve protein production in CHO cells. Thus, the aim of the present study was to examine if miR-143 might be suitable to improve production of low yield protein candidates. Both transient and stable overexpression of miR-143 significantly improved protein production without negatively affecting cell growth and viability of different recombinant CHO cells. In addition, mitogen-activated protein kinase 7 (MAPK7) was identified as a putative target gene of miR-143-3p in CHO cells. Finally, siRNA-mediated knock-down of MAPK7 could be demonstrated to phenocopy pro-productive effects of miR-143. In summary, our data suggest that miR-143 might represent a novel genetic element to enhance production of difficult-to-express proteins in CHO cells which may be partly mediated by down-regulation of MAPK7. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1046-1058, 2017.

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“…The majority of studies into miRs in CHO to date have focused upon profiling or manipulation of miR amounts (e.g., refs. ), but here we focused upon utilizing endogenous amounts of highly expressed and conserved miRs. Selecting and exploiting the endogenous miR profile of the cell means that miR mediated repression would in theory not require the manipulation of the miR itself which could result in unwanted side‐effects in terms of changing the gene expression of the CHO host.…”

Section: Discussionmentioning

confidence: 99%

Application of microRNA Targeted 3′UTRs to Repress DHFR Selection Marker Expression for Development of Recombinant Antibody Expressing CHO Cell Pools

Jossé

Zhang

Smales

2018

Biotechnology Journal

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The dihydrofolate reductase (DHFR) system is used for the selection of recombinant Chinese hamster ovary (CHO) cell lines using the inhibitor methotrexate (MTX). During clonal selection, endogenous DHFR expression, and resistance to MTX allows the selection of cells expressing sufficient DHFR to survive. Here, the authors describe a novel vector platform for the DHFR system, whereby addition of a synthetic 3'UTR destabilizes DHFR expression. miRs ability to negatively regulate gene expression by their near-complementary binding to the 3'UTR region of transcripts are harnessed. From the literature, the authors identified let-7f as a highly abundant, invariant miR in CHO cells. Three 3'UTR targets of the let-7f miR are then cloned in the DHFR host 3'UTR to determine the impact on gene expression (HMGA2 3'UTR sequence 1, 2, and 3). Using luciferase as a reporter, the authors show down-regulation of luciferase activity is mediated by the nature of the 3'UTR and its ability to bind let-7f. The same 3'UTRs downstream of the DHFR gene to show this also results in reduced transcript amounts are then applied. Finally, the authors applied this methodology to generate stable DG44-derived cell pools expressing a model monoclonal antibody (mAb), demonstrating this approach can be used for the selection of antibody-producing cells with low MTX concentrations.

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miRNA profiling of high, low and non-producing CHO cells during biphasic fed-batch cultivation reveals process relevant targets for host cell engineering

Cited by 23 publications

References 66 publications

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

miR‐143 targets MAPK7 in CHO cells and induces a hyperproductive phenotype to enhance production of difficult‐to‐express proteins

Application of microRNA Targeted 3′UTRs to Repress DHFR Selection Marker Expression for Development of Recombinant Antibody Expressing CHO Cell Pools

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