Recent advances in genome engineering technologies have enabled targeted manipulation of the CHO cell for enhanced process performance. However, the engineered cells invariably undergo clonal isolation, and thus, potential clonal variability poses concerns on the utility of genome engineering. Significant clonal variability can potentially diminish the effects of targeted cell engineering efforts.We examined the clonal variation in recombinant protein producing CHO cells. Single cell clones were isolated from a stable high (IgG) producing cell line which had been extensively cultured. High-producing and low-producing sub-clones were isolated and then, individually sub-cloned further. To elucidate the root cause of the observed clonal variation, we examined the transcriptome of these subclones. Variability is seen not only at the productivity and growth rate level, but also at the expression of genes of various functional classes.  Using NextGen sequencing on the genome we also estimated the mutation frequency in CHO cells. The results indicate that the accumulation of mutation in the population is not at an exceedingly high level, thus suggesting clonal variations may arise from other events such as epigenetic changes. We also conducted sequencing to identify the transgene integration sites for an analysis of clonal stability in a genomic context.  Clonal variation is a significant factor of consideration in evaluating the suitability of  targeted transgene integration for constructing producing cell lines. Our results suggest that variability may arise after clonal selection. To ensure a high rate of success a probabilistic model for clonal isolation is possibly warranted.

Clonal stability considerations in targeted genome engineering

247th National Meeting (2014)

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