Single‐cell RNA sequencing reveals population‐based shifts in gene expression heterogeneity in an unstable, clonally derived CHO cell line

The genomic plasticity of Chinese Hamster Ovary cells allows them to be genetically engineered to produce foreign proteins with high specific productivities while placing a significant burden on their ability to retain this expression capacity over time. Many cell lines lose expression over time and need to be screened for expression consistency. Stability has been studied in clonally derived cell cultures by evaluating phenotypic heterogeneity in subclones derived from stable or unstable clones, but not well characterized within the primary, clonally derived cell culture. To address this, we employed single cell RNA sequencing in stable and unstable cell lines expressing the same monoclonal antibody, at early and late timepoints in their manufacturing lifespan. We observed higher expression heterogeneity and a more drastic redistribution of expression profile "clusters" over serial passaging in the unstable cell line. Differentially expressed genes in those clusters making up the unstable cell line suggest distinct changes in metabolic pathway activity in this cell line over time. Using molecular methods, changing gene expression profiles were confirmed and activation of molecular pathways consistent with a challenged protein homeostasis were shown to be enriched in cells over time in the unstable cell line. This report more broadly implicates the role of heterogeneity within a clonal population as a contributing factor to cell line instability, providing a biological context for further investigation.