Single-cell RNA sequencing reveals homogeneous transcriptome patterns and low variance in a suspension CHO-K1 and an adherent HEK293FT cell line in culture conditions

Borsi, Giulia; Motheramgari, Krishna; Dhiman, Heena; Baumann, Martina; Sinkala, Elly; Sauerland, Max; Riba, Julian; Borth, Nicole

doi:10.1016/j.jbiotec.2023.01.006

Cited by 7 publications

(5 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our results show that overall transcriptional diversity within the population is low with no distinct subpopulation present at any time during the CLD process, as previously shown for CHO and HEK cells 33 . In contrast, transgene expression shows a high degree of variability within the host (originally clonal, but passaged over > 6 months) and a stable pool population despite the use of an isogenic, single-copy targeted integration method.…”

Section: Discussionsupporting

confidence: 87%

“…In establishing and validating this method, we have focused on a state-of-the-art targeted integration CLD workflow for CHO-cell biofactories producing a panel of three distinct complex antibodies. www.nature.com/scientificreports/ Our results show that overall transcriptional diversity within the population is low with no distinct subpopulation present at any time during the CLD process, as previously shown for CHO and HEK cells 33 . In contrast, transgene expression shows a high degree of variability within the host (originally clonal, but passaged over > 6 months) and a stable pool population despite the use of an isogenic, single-copy targeted integration method.…”

Section: Discussionsupporting

confidence: 84%

See 1 more Smart Citation

Genomic barcoding for clonal diversity monitoring and control in cell-based complex antibody production

Bauer,

Oberist,

Poth

et al. 2024

Sci Rep

View full text Add to dashboard Cite

Engineered mammalian cells are key for biotechnology by enabling broad applications ranging from in vitro model systems to therapeutic biofactories. Engineered cell lines exist as a population containing sub-lineages of cell clones that exhibit substantial genetic and phenotypic heterogeneity. There is still a limited understanding of the source of this inter-clonal heterogeneity as well as its implications for biotechnological applications. Here, we developed a genomic barcoding strategy for a targeted integration (TI)-based CHO antibody producer cell line development process. This technology provided novel insights about clone diversity during stable cell line selection on pool level, enabled an imaging-independent monoclonality assessment after single cell cloning, and eventually improved hit-picking of antibody producer clones by monitoring of cellular lineages during the cell line development (CLD) process. Specifically, we observed that CHO producer pools generated by TI of two plasmids at a single genomic site displayed a low diversity (< 0.1% RMCE efficiency), which further depends on the expressed molecules, and underwent rapid population skewing towards dominant clones during routine cultivation. Clonal cell lines from one individual TI event demonstrated a significantly lower variance regarding production-relevant and phenotypic parameters as compared to cell lines from distinct TI events. This implies that the observed cellular diversity lies within pre-existing cell-intrinsic factors and that the majority of clonal variation did not develop during the CLD process, especially during single cell cloning. Using cellular barcodes as a proxy for cellular diversity, we improved our CLD screening workflow and enriched diversity of production-relevant parameters substantially. This work, by enabling clonal diversity monitoring and control, paves the way for an economically valuable and data-driven CLD process.

show abstract

Section: Discussionsupporting

confidence: 87%

Section: Discussionsupporting

confidence: 84%

Genomic barcoding for clonal diversity monitoring and control in cell-based complex antibody production

Bauer,

Oberist,

Poth

et al. 2024

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Although non-human mammalian cells have safety and efficacy, RTPs expressed are easy to cause immunity in the human body, resulting in the elimination of foreign RTPs by the circulatory system, due to the different PTMs, especially the difference in glycosylation from human cells. Human embryonic kidney (HEK) 293 cells are widely used as human cell lines for recombinant protein drug production (Borsi et al 2023 ). Mammalian cell lines have the integrated capabilities to express and secrete RTPs, and a large number of cell lines with suitable growth characteristics that could be obtained from various tissues and species.…”

Section: Introductionmentioning

confidence: 99%

Recombinant therapeutic proteins degradation and overcoming strategies in CHO cells

Geng,

Zhao,

Zhang

et al. 2024

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

Mammalian cell lines are frequently used as the preferred host cells for producing recombinant therapeutic proteins (RTPs) having post-translational modified modification similar to those observed in proteins produced by human cells. Nowadays, most RTPs approved for marketing are produced in Chinese hamster ovary (CHO) cells. Recombinant therapeutic antibodies are among the most important and promising RTPs for biomedical applications. One of the issues that occurs during development of RTPs is their degradation, which caused by a variety of factors and reducing quality of RTPs. RTP degradation is especially concerning as they could result in reduced biological functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and generate potentially immunogenic species. Therefore, the mechanisms underlying RTP degradation and strategies for avoiding degradation have regained an interest from academia and industry. In this review, we outline recent progress in this field, with a focus on factors that cause degradation during RTP production and the development of strategies for overcoming RTP degradation. Key points • The recombinant therapeutic protein degradation in CHO cell systems is reviewed. • Enzymatic factors and non-enzymatic methods influence recombinant therapeutic protein degradation. • Reducing the degradation can improve the quality of recombinant therapeutic proteins.

show abstract

“…They did not observe other clear substructures, though they did see significant variation in enolase expression within their clusters. Other recent work such as Borsi et al (2023) compared the transcriptional profiles of differently aged CHO-K1 vs HEK293FT cell lines, specifically looking for highly variable genes shared between the two cell lines (Borsi et al, 2023). Borsi et al identified 53 genes that defined the transcriptomic variability in both the HEK and CHO cell lines.…”

Section: Introductionmentioning

confidence: 99%

“…But the real concern is not just understanding a snapshot of heterogeneity (Tzani et al, 2021) nor how cells perform over a limited span (Borsi et al, 2023; Ogata et al, 2021) but rather why production drops over time . To accomplish this we used 10X Chromium single-cell sequencing to examine the transcriptome of two CHO clones over 90 days (30 population doublings).…”

Section: Introductionmentioning

confidence: 99%

Investigating Subpopulation Dynamics in Clonal CHO-K1 Cells with Single-Cell RNA Sequencing

Morina,

Cao,

Chen

et al. 2024

Preprint

View full text Add to dashboard Cite

Chinese Hamster Ovary (CHO) cells are used to produce monoclonal antibodies and other biotherapeutics at industrial scale. Despite their ubiquitous nature in the biopharmaceutical industry, little is known about the behaviors of individual transfected clonal CHO cells. Most CHO cells are assessed on their ability to produce the protein of interest over time, known as their stability. But these CHO cells have primarily been studied in bulk, working under the assumption that these bulk samples are identical because of genetic clonality across the sample; however, this does not address other forms of cellular heterogeneity in these ostensibly clonal cells. It is possible these variable stability phenotypes reflect heterogeneity within the clonal samples. In this study, we performed single-cell RNA sequencing on two clonal CHO-K1 cell populations with different stability phenotypes over a 90 day culture period. Our data showed that the instability of the unstable clone was due in part to the emergence of a low-producing subpopulation in the aged samples. This low-producing subpopulation did not exhibit markers of cellular stress which were expressed in the higher-producing populations. Further multiomic investigation should be performed to better characterize this heterogeneity.

show abstract

Single-cell RNA sequencing reveals homogeneous transcriptome patterns and low variance in a suspension CHO-K1 and an adherent HEK293FT cell line in culture conditions

Cited by 7 publications

References 64 publications

Genomic barcoding for clonal diversity monitoring and control in cell-based complex antibody production

Genomic barcoding for clonal diversity monitoring and control in cell-based complex antibody production

Recombinant therapeutic proteins degradation and overcoming strategies in CHO cells

Investigating Subpopulation Dynamics in Clonal CHO-K1 Cells with Single-Cell RNA Sequencing

Contact Info

Product

Resources

About