Subcloning induces changes in the DNA‐methylation pattern of outgrowing Chinese hamster ovary cell colonies

Abstract

Chinese hamster ovary (CHO) cells are the most extensively used mammalian production system for biologics intended for use in humans. A critical step in the establishment of production cell lines is single cell cloning, with the objective of achieving high productivity and product quality. Despite general use, knowledge of the effects of this process is limited. Importantly, single cell cloned cells display a wide array of observed phenotypes, which so far was attributed to the instability and variability of the CHO genome. In this study we present data indicating that the emergence of diverse phenotypes during single cell cloning is associated with changes in DNA methylation patterns and transcriptomes that occur during the subcloning process. The DNA methylation pattern of each analyzed subclone, randomly picked from all outgrowing clones of the experiment, had unique changes preferentially found in regulatory regions of the genome such as enhancers, and de‐enriched in actively transcribed sequences (not including the respective promoters), indicating that these changes resulted in adaptations of the relative gene expression pattern. The transcriptome of each subclone also had a significant number of individual changes. These results indicate that epigenetic regulation is a hidden, but important player in cell line development with a major role in the establishment of high performing clones with improved characteristics for bioprocessing.

Metabolic Profiling of CHO Cells during the Production of Biotherapeutics

“…These discrepancies may be explained by the unique features of the CHO cell lines derived from a single host, acquired by genetic drift in separate laboratories [ 60 ]. Differences in terms of the media used and the culture strategies have also shown to give rise to genetic and epigenetic diversity, and this could explain phenotypic differences when distinct processes are used for the production from the same clone [ 61 , 62 ]. This diversity is known to have a major impact on the metabolomic profile of CHO culture.…”

Improved Titer in Late-Stage Mammalian Cell Culture Manufacturing by Re-Cloning

“…This heterogeneity is generally accepted in the field and, importantly, is not indicative of instability in upstream process performance or product quality [6,23,37,38]. More recently, it has been suggested that the process of single cell cloning itself can impact CHO cell epigenetics in a heritable manner, leading to altered (and possibly improved) performance [39]. The data presented in this report would be consistent with both hypotheses, and it is possible that both heterogeneity of the parental culture and epigenetic change in the re-clones play a role in the titer improvement that was observed in the re-clones.…”

Spotlight on Three Rs Progress

“…Subclones can exhibit a wide range of phenotypes, even when derived from a subcloned parent population. Weinguny et al 1 set out to investigate the role of epigenetic modifications — specifically DNA methylation — on the diversity of individual subclones of CHO cells. The effects of such modifications can be passed on to daughter cells.…”

“…lack of contact with other cells) during the subcloning process. The authors conclude that: “This process appears to be caused by the cells activating a yet unknown mechanism to actively and randomly change their DNA methylation pattern, resulting in new and unique patterns that were not present in the parental population.” 1…”