Growth Rate Changes in CHO Host Cells Are Associated with Karyotypic Heterogeneity

Abstract

Chinese hamster ovary (CHO) cell line instability and clonality issues can affect cell culture phenotypes such as cell growth, productivity, or product quality and remain challenges for biopharmaceutical manufacturing. While there have been efforts for characterizing cell line instability in CHO production cell lines, a pre-existing level of cell line instability in CHO host cells has not been determined. In this study, cell line instability and chromosomal heterogeneity of the host, CHO-DUK cell line, is reported by using a karyotyping approach. Long-term cultures and karyotype analysis of CHO-DUK cells revealed that the growth rate was higher in later passage cultures, correlating with an increase in the population ratio containing the mar3 chromosome. To further investigate a correlation between growth rate and karyotype, CHO-DUK cells are subcloned by limiting dilution and the growth rate and karyotype of each subclone are determined. Subclones containing the mar3 chromosome exhibit higher cell growth rates than subclones without the mar3 chromosome. Finally, karyotype analysis indicate that CHO-DUK cells, as well as limiting-diluted subclones, exhibit a karyotypically heterogeneous population, suggesting that chromosomal rearrangements occur spontaneously and frequently even in non-engineered host cells. These results demonstrate CHO host cell line instability and suggest that chromosomal instability and karyotypic changes are associated with compromised clonality (heterogeneity), affecting cell line (in)stability in CHO host cells.

Characterization of phenotypic and genotypic diversity in subclones derived from a clonal cell line

“…Cells in culture, especially immortalized cell lines such as CHO, display well‐documented genetic plasticity, which when coupled with the large number of population doublings from the cloning stage can result in phenotypic and genotypic differences over time . These changes in phenotype have been linked to environmental influence, epigenetic silencing, chromosomal instability, and karyotypic changes . Given the number of population doublings that occur from single cell plating to a production bioreactor and the known plasticity observed in CHO cells, genetic and epigenetic drift can occur through the sub culturing and adaption phases during clonal outgrowth and subsequent culture during banking and bioprocess …”

Cell function profiling to assess clone stability

“…Chinese hamster ovary (CHO) cells remain the major mammalian platform for biopharmaceutical production despite a propensity for rapid change (Baik & Lee, 2018; Barnes, Bentley, & Dickson, 2003; Davies et al, 2013; Fernandez‐Martell, Johari, & James, 2018; O'Callaghan et al, 2010) that can render a bioprocess uneconomic or generate product deviations incompatible with the clinic. During cell line development (CLD) companies are required to demonstrate production clone stability by monitoring changes to titer, product quality, and other key performance indicators over prolonged subculture (ICH Expert Working Group, 1997).…”

CRISPR‐Based Targeted Epigenetic Editing Enables Gene Expression Modulation of the Silenced Beta‐Galactoside Alpha‐2,6‐Sialyltransferase 1 in CHO Cells

“…Growth and recombinant protein productivity was not affected upon activation of ST6GAL1, in contrast to applications where traditional genetic engineering approaches are used, which require subcloning to isolate the few cells that have integrated the recombinant gene . These approaches also require the use of selective markers, where their expression will increase the metabolic burden on cells and consequently affect cell behavior.…”