Investigating subpopulation dynamics in clonal CHO-K1 cells with single-cell RNA sequencing

Chinese Hamster Ovary (CHO) cells 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 stability, their ability to produce the protein of interest over time. But CHO cells have primarily been studied in bulk, instead assuming that these bulk samples are homogenous because of presumed genetic clonality across the sample. This does not address cellular heterogeneity in these ostensibly clonal cells. These variable stability phenotypes may 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 one of the clone’s gene expression 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. •Generated transcriptomic data from >5000 cells across two clones over 90 days to analyze cell heterogeneity.•Transcriptional heterogeneity within clonal populations contributes to decreased cell productivity over time.•Identified high/low productivity cell clusters with distinct marker genes beyond target genes, including Scd2.•In silico marker-based filtering could eliminate low-producing cell subpopulations.