Antibody expression stability in CHO clonally derived cell lines and their subclones: Role of methylation in phenotypic and epigenetic heterogeneity

Routine CHO cell line development practices involve a lengthy process of iteratively screening clonally derived cell lines to identify a single line suitable for IND filing and clinical manufacture. Paramount in this process is development of a stable production cell line having consistent growth, productivity and product quality for the entire generational length of the manufacturing process. Scale-down stability models used to screen clones for consistency are time consuming and often a rate-limiting step in clone selection. To investigate CHEF1 production stability in CHO cells we analyzed genotypic and phenotypic attributes of monoclonal primary clones and their respective subclones over time in standard antibody production models. The main finding of this work indicates that monoclonal cell lines derived from single cell progenitors grow into populations of cells with varied phenotypic heterogeneity, as revealed in their subclones, from either stable or unstable cell lines. Investigation of the subclones demonstrates that clonally derived cell lines grow out into populations with variable phenotypes and genotypes, even if the primary clone shows consistency in both over many generations in a stability study. Phenotypic and genotypic heterogeneity mostly did not correlate, but growth and productivity appear driven in part by cytosine methylation heterogeneity in both primary and secondary clones. This work presents evidence that epigenetic analysis may be useful for early detection of stability traits, but emphasizes the continued importance of rigorous cell line stability screening to identify primary clones that have consistent phenotypic characteristics, especially growth and productivity, throughout the in vitro lifecycle of the cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:635-649, 2018.