Tracing production instability in a clonally derived CHO cell line using single-cell transcriptomics
| Autor: | Colin Clarke, Niall Barron, Jonathan Bones, Sara Carillo, Cathy A Spargo, W Shannon Dillmore, Ioanna Tzani, Ryan Hagan, Nicholas Herrmann |
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| Rok vydání: | 2021 |
| Předmět: |
Transgene
Cell Population Bioengineering CHO Cells Biology Immunoglobulin light chain Applied Microbiology and Biotechnology Transcriptome 03 medical and health sciences 0302 clinical medicine Cricetulus Cricetinae medicine Gene silencing Animals Transgenes education Gene 030304 developmental biology 0303 health sciences education.field_of_study Chinese hamster ovary cell Gene Expression Profiling Antibodies Monoclonal High-Throughput Nucleotide Sequencing Cell biology medicine.anatomical_structure Cell culture Single-Cell Analysis 030217 neurology & neurosurgery Biotechnology |
| Zdroj: | Biotechnology and Bioengineering |
| ISSN: | 1097-0290 |
| Popis: | A variety of mechanisms including transcriptional silencing, gene copy loss and increased susceptibility to cellular stress have been associated with a sudden or gradual loss of monoclonal antibody (mAb) production in Chinese hamster ovary (CHO) cell lines. In this study, we utilised single cell RNA-seq (scRNA-seq) to study a clonally-derived CHO cell line that underwent production instability leading to a dramatic reduction of the levels of mAb produced. From the scRNA-seq data we identified sub clusters associated with variations in the mAb transgenes and observed that heavy chain gene expression was significantly lower than that of the light chain across the population. Using trajectory inference, the evolution of the cell line was reconstructed and was found to correlate with a reduction in heavy and light chain gene expression. Genes encoding for proteins involved in the response to oxidative stress and apoptosis were found to increase in expression as cells progressed along the trajectory. Future studies of CHO cell lines using this technology have the potential to dramatically enhance our understanding of the characteristics underpinning efficient manufacturing performance as well as product quality.HighlightsA clonally-derived CHO cell line in our laboratory had undergone production instability – in that the amount of intact monoclonal antibody had reduced dramatically to levels at which reliable quantitation was no longer possible. We were, however, able to detect mAb heavy and light chain protein, as well as dimerised light chain species in the cell culture media.Single cell RNA-seq was utilised to capture > 3,800 gene expression profiles from the cell line at 72hrs post seeding.Analyses of the scRNA-seq data uncovered transcriptional heterogeneity and revealed the presence of multiple intra cell line clusters. The heavy chain transcript was detected at a significantly lower level in comparison to light chain transcripts. Light chain gene expression was not only more abundant, but also expressed more uniformly across the cell population.Using unsupervised trajectory analysis, the emergence of heterogeneity in the cell population was traced from those cells most similar to the original isolated clone to those where transcription of the mAb heavy and light chain was undetectable.Subsequent analysis of CHO cell gene expression patterns revealed a correlation between the progression of cells along the trajectory and the upregulation of genes involved in the cellular response to oxidative stress. |
| Databáze: | OpenAIRE |
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