Promoter methylation and transgene copy numbers predict unstable protein production in recombinant chinese hamster ovary cell lines

Abstract

Mammalian cell lines for recombinant protein production need to maintain productivity over extended cultivation times. Long-term stability studies are time and resource intensive, but are widely performed to identify and eliminate unstable candidates during cell line development. Production instability of manufacturing cell lines can be associated with methylation and silencing of the heterologous promoter. We have identified CpG dinucleotides within the human cytomegalovirus major immediate early promoter/enhancer (hCMV-MIE) that are frequently methylated in unstable antibody-producing Chinese hamster ovary (CHO) cell lines. We have established methylation-specific real-time qPCR for the rapid and sensitive measurement of hCMV-MIE methylation in multiple cell lines and provide evidence that hCMV-MIE methylation and transgene copy numbers can be used as early markers to predict production instability of recombinant CHO cell lines. These markers should provide the opportunity to enrich stable producers early in cell line development and allow developers to put more emphasis on other criteria, such as product quality and bioprocess robustness.

Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus

“…8); other possible mechanisms such as changes of status in histone acetylation between the insulated and uninsulated clones after differentiation will be examined. Our system, with the ability to target only one copy of transgene at a defined genomic locus, has provided a platform allowing for further investigation along this line, since uncontrolled transgene copy number has also been reported to interfere with epigenetic modifications [32]. Additional experiments to investigate the comprehensive mechanisms that different types of chromatin insulators establish to shield transgenes from silencing in hPSCs are of interest.…”

Building a Cell Culture Process with Stable Foundations: Searching for Certainty in an Uncertain World

“…However, the relative importance of gene loss verses transcriptional silencing in specific cell lines may depend on the host as well as the gene expression system used (Kim et al 2011). Interestingly, in some cases both gene loss and silencing may occur (Osterlehner et al 2011). Loss of productivity caused by epigenetic gene silencing is specifically recognised when the loss of qP is not associated with a loss of recombinant gene copies but with a decline in recombinant mRNA content.…”

“…This threshold level is thought to be the point at which the downstream biosynthetic reactions utilising recombinant mRNAs are saturated, and therefore only when the transcript copy number declines further will there be any phenotypic consequence. Cell line instability can be observed both in the presence and absence of selective pressure, although it may be both more likely and more precipitous once the selective pressure has been removed (Dorai et al 2012;Kim et al 2011;Osterlehner et al 2011;Yang et al 2010;Chusainow et al 2009). Where a decline in recombinant Mab mRNA has been observed, it can affect both the HC and LC genes in cell lines created using both DHFR and GS expression systems, as well as in low and high productivity clones (Kim et al 2011;Yang et al 2010;Chusainow et al 2009).…”

“…In particular, Osterlehner and colleagues identified a specific CpG site (À179 relative to the TSS) whose methylation status at early phase culture was predictive of the stability profile of 75 % of clones over long-term sub-culture, although recombinant mRNA content was not measured in this study. Unstable clones also tended to have higher transgene copy number, perhaps suggesting the involvement of tandem repeat silencing by methylation as an underlying mechanism in the cell lines studied (Osterlehner et al 2011). Based on these findings the authors suggest that clones should be selected on the basis of low transgene copy number to minimise the chance of instability later in culture.…”

“…As observed by Kim et al (2011), the hCMV-MIE contains a CpG island consisting of 29 CpG sites, the vast majority of which are known methylation targets. Several studies have observed an increase in hCMV promoter methylation across long-term sub-culture correlating with a decrease in productivity and a decline in recombinant mRNA copy number (Kim et al 2011;Osterlehner et al 2011;Yang et al 2010;Chusainow et al 2009). Kim et al (2011) demonstrated that loss of qP with increasing number of population doublings in recombinant Mab-producing GS-CHO cells was associated with a decline in Mab HC and LC mRNA content and an increase in hCMV-MIE methylation of the promoter CpG island.…”