Directed evolution approach to enhance efficiency and speed of outgrowth during single cell subcloning of Chinese Hamster Ovary cells

Weinguny, Marcus; Klanert, Gerald; Eisenhut, Peter; Jonsson, Andreas; Ivansson, Daniel; Lövgren, Ann; Borth, Nicole

doi:10.1016/j.csbj.2020.05.020

Cited by 17 publications

(19 citation statements)

References 60 publications

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“…A recent study revealed that a major contributor to this stress is the absence of cell‐to‐cell communication and, possibly, the lack of proteins and other cellular signals in the culture environment. [ 59 ] CHO cells come from an organism where they are used to being in constant contact and communication with other cells and tissues, and where a continuous network of signals is being transmitted to stimulate and support them. [ 50 ] In the usual suspension culture environment where cells are present in large number, this communication network is generated by the cells themselves who secrete proteins and other communication tools such as exosomes.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…They also align well with the pathways identified in a recent study to support more efficient clonal outgrowth in cell lines selected for this property. [ 59 ] Of interest, in view of the know effect of subcloning to stabilize and “rejuvenate” cells, is the differential regulation of pathways and genes related to DNA repair.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, we suggest that our results support the hypothesis that these new methylation patterns were actively generated by the cells in response to the stress caused by isolation during the subcloning process itself. [59] An important question in this context is at what stage such a process of re-methylation takes place and for how long it lasts. In the new subclones, a notable part of the new methylation pattern was again uniform throughout the population, with 3%-9% fully methylated or non-methylated.…”

Section: Discussionmentioning

confidence: 99%

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Subcloning induces changes in the DNA‐methylation pattern of outgrowing Chinese hamster ovary cell colonies

Weinguny

Klanert

Eisenhut

et al. 2021

Biotechnology Journal

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Chinese hamster ovary (CHO) cells are the most extensively used mammalian production system for biologics intended for use in humans. A critical step in the establishment of production cell lines is single cell cloning, with the objective of achieving high productivity and product quality. Despite general use, knowledge of the effects of this process is limited. Importantly, single cell cloned cells display a wide array of observed phenotypes, which so far was attributed to the instability and variability of the CHO genome. In this study we present data indicating that the emergence of diverse phenotypes during single cell cloning is associated with changes in DNA methylation patterns and transcriptomes that occur during the subcloning process. The DNA methylation pattern of each analyzed subclone, randomly picked from all outgrowing clones of the experiment, had unique changes preferentially found in regulatory regions of the genome such as enhancers, and de‐enriched in actively transcribed sequences (not including the respective promoters), indicating that these changes resulted in adaptations of the relative gene expression pattern. The transcriptome of each subclone also had a significant number of individual changes. These results indicate that epigenetic regulation is a hidden, but important player in cell line development with a major role in the establishment of high performing clones with improved characteristics for bioprocessing.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Subcloning induces changes in the DNA‐methylation pattern of outgrowing Chinese hamster ovary cell colonies

Weinguny

Klanert

Eisenhut

et al. 2021

Biotechnology Journal

Self Cite

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show abstract

“… 15 Improved subcloning performance and enhanced growth behavior were reported using single-cell sorting as the evolutionary pressure. 16 Adaptation of an IgG-producing CHO cell line to endoplasmic reticulum (ER) stress by continuous culturing under increasing concentration of tunicamycin resulted in higher productivity due to increased ER capacity. 17 Recently, Mistry et al described evolution of CHO host cells in the presence of hydrogen peroxide to generate oxidative stress-resistant host cells that showed improved expression of difficult-to-express (DTE) proteins.…”

Section: Introductionmentioning

confidence: 99%

Mitochondrial membrane potential-enriched CHO host: a novel and powerful tool for improving biomanufacturing capability

Chakrabarti

Chaerkady

Wang

et al. 2022

mAbs

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With the aim of increasing protein productivity of Chinese hamster ovary (CHO) cells, we sought to generate new CHO hosts with favorable biomanufacturing phenotypes and improved functionality. Here, we present an innovative approach of enriching the CHO host cells with a high mitochondrial membrane potential (MMP). Stable transfectant pools and clonal cell lines expressing difficult-to-express bispecific molecules generated from the MMP-enriched host outperformed the parental host by displaying (1) improved fed-batch productivity; (2) enhanced long-term cell viability of pools; (3) more favorable lactate metabolism; and (4) improved cell cloning efficiency during monoclonal cell line generation. Proteomic analysis together with Western blot validation were used to investigate the underlying mechanisms by which high MMP influenced production performance. The MMP-enriched host exhibited multifaceted protection against mitochondrial dysfunction and endoplasmic reticulum stress. Our findings indicate that the MMP-enriched host achieved an overall “fitter” phenotype that contributes to the significant improvement in biomanufacturing capability.

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Naming CHO cells for bio‐manufacturing: Genome plasticity and variant phenotypes of cell populations in bioreactors question the relevance of old names

Wurm

2021

Biotechnology Journal

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Chinese Hamster Ovary [CHO] cells are the workhorse for production of modern biopharmaceuticals. They are however immortalized cells with a high propensity for genetic change. Judging from published culture records, CHO cell populations have undergone hundreds of population doublings since their origin in the late 1950s. Different cell populations were established and named from 1 to 3 decades after their generation, such as CHO‐Pro–, CHO‐K1, CHO‐DG44, CHO‐S, CHO‐DUK, CHO‐DXB‐11 to indicate origin and certain phenotypic features. These names are commonly used in scientific publications still today. This article discusses the relevance of such names. We argue that they provide a false sense of identity. To substantiate this, we provide the long (and poorly recorded) history of CHO cells as well as their highly complex genetics. Finally, we suggest an alternative naming system for CHO cells which provides more relevant information. While the implementation of a new naming convention will require substantial discussions among members of the relevant community, it should improve interpretation and comparability between laboratories. This, in turn will help scientific communities and industrial users to attain and further the full potential of CHO cells.

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Directed evolution approach to enhance efficiency and speed of outgrowth during single cell subcloning of Chinese Hamster Ovary cells

Cited by 17 publications

References 60 publications

Subcloning induces changes in the DNA‐methylation pattern of outgrowing Chinese hamster ovary cell colonies

Subcloning induces changes in the DNA‐methylation pattern of outgrowing Chinese hamster ovary cell colonies

Mitochondrial membrane potential-enriched CHO host: a novel and powerful tool for improving biomanufacturing capability

Naming CHO cells for bio‐manufacturing: Genome plasticity and variant phenotypes of cell populations in bioreactors question the relevance of old names

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