Recurring genomic structural variation leads to clonal instability and loss of productivity

Abstract

Chinese hamster ovary cells, commonly used in the production of therapeutic proteins, are aneuploid. Their chromosomes bear structural abnormality and undergo changes in structure and number during cell proliferation. Some production cell lines are unstable and lose their productivity over time in the manufacturing process and during the product's life cycle. To better understand the link between genomic structural changes and productivity stability, an immunoglobulin G producing cell line was successively single-cell cloned to obtain subclones that retained or lost productivity, and their genomic features were compared. Although each subclone started with a single karyotype, the progeny quickly diversified to a population with a distribution of chromosome numbers that is not distinctive from the parent and among subclones. The comparative genomic hybridization (CGH) analysis showed that the extent of copy variation of gene coding regions among different subclones stayed at levels of a few percent. Genome regions that were prone to loss of copies, including one with a product transgene integration site, were identified in CGH. The loss of the transgene copy was accompanied by loss of transgene transcript level. Sequence analysis of the host cell and parental producing cell showed prominent structural variations within the regions prone to loss of copies. Taken together, we demonstrated the transient nature of clonal homogeneity in cell line development and the retention of a population distribution of chromosome numbers; we further demonstrated that structural variation in the transgene integration region caused cell line instability. Future cell line development may target the transgene into structurally stable regions.

Characterization of metabolic responses, genetic variations, and microsatellite instability in ammonia-stressed CHO cells grown in fed-batch cultures

“…Previous studies have characterized genomic variants across various CHO cell lines that may be a contributing factor to genome instability [ 9 – 11 ]. An unstable genome can result in reduced productivity in continuous cultures and fed-batch systems [ 12 , 13 ]. A common occurrence in both continuous cultures and fed-batch systems is the accumulation of metabolic waste products, such as ammonia and lactate.…”

Chinese hamster ovary cell line DXB-11: chromosomal instability and karyotype heterogeneity

“…The undoubted advantages of immortalized CHO cells include a high proliferation rate, and the ability to adapt to genetic manipulations and culture conditions. At the same time, these properties are associated with an increased mutation rate, changes in the genome structure and DNA methylation pattern [ 7 – 9 ]. The karyotype heterogeneity of CHO cells increases with prolonged cultivation [ 10 ] and is inevitably reproduced in course of subcloning of cells [ 11 – 13 ].…”

HIF‐1 Signaling Pathway Implicated in Phenotypic Instability in a Chinese Hamster Ovary Production Cell Line

“…CHO cell line instability and its impact on cell line development remain a great challenge. In contrast to the genotypic instability for which the causes at the molecular level such as transgene loss or epigenetic regulations are well‐studied and characterized, very few studies have been reported on phenotypic instability. In this study, two clonal cell lines derived from Pfizer's SSI expression system were examined; both cell lines demonstrated genotypic stability, yet one exhibited an age‐related phenotypic instability (Cell Line A) and the other (Cell Line B) did not.…”

“…Loss of expression in recombinant CHO cells has been extensively studied, and many groups have reported the loss of transgene copies or transcriptional silencing of promoters as the main causes. In contrast, there is minimal literature available on phenotypic instability.…”