Single-Cell Analysis of Chinese Hamster Ovary Mitochondrial DNA

The vast majority of recombinant therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cells. However, it is still recognised that genetic and phenotypic heterogeneity arises over time. The key role of energy for recombinant protein production has led teams to focus on the mitochondria; which contain their own genome in the form of mitochondrial DNA (mtDNA). Previous bulk analysis of CHO cell populations revealed considerable mtDNA variation (heteroplasmy) which could contribute to the metabolic variability often observed. However, bulk analysis neglects the differences between individual cells within the population. By analysing the mtDNA differences of single-cells, this heterogeneity can be characterised with greater specificity. Here, we developed a protocol to sequence single CHO cells from cell culture through to bioinformatic analysis. Then, we expanded our analysis to 84 single CHO cells. We also exploited scRNAseq for multi-modal analysis of mtDNA from thousands of single CHO cells. We observed great variability in allele frequency among single-cells, with possible contributions to phenotypic changes. We attempted to apply our method to single mitochondria but were not able to amplify from the tiny starting mass of DNA. We also used our scRNAseq datasets to compare CHO cells in “high-producing” and “low-producing” populations. Previous scRNAseq analysis of “instability” focused on a mAb-producing CHO cell line which progressively lost productivity over time. However, comparisons were hindered by the comparatively longer time the “low-producing” CHO cell population spent in culture. Here, we exploited the inducible nature of the CHO-PVZ and CHO-RTX cell lines. When cumate was added, mAb production was “switched-on” leading to over 6x greater titre after 3 days in both cell lines; while maintaining control of other environmental factors. We generated lists of genes whose expression was impacted upon induction of antibody expression, finding 18 that were common to both cell lines. This list represents genes whose expression is impacted by or supports the process of producing an antibody but not specific to the mAb being produced. Gene ontology tests converged to suggest a key role of the Unfolded Protein Response and Endosomal Reticulum Associated Degradation pathways in high-producing CHO cell lines.