CHO cells are the most frequently used host for commercial production of therapeutic proteins, and DHFR-mediated gene amplification is extensively applied to generate cell lines with increased protein production. However, decreased protein productivity is observed unpredictably during the time required for scale-up with consequences for yield, time, finance and regulatory approval. In this study, we have examined the interaction between Ubiquitous Chromatin Opening Elements (UCOE) and DHFR-linked amplification in relation to cell expression stability. In summary, the inclusion of UCOE elements generated cells that (1) achieved higher cell densities and exhibited increased production of recombinant mRNA per cell and protein yield, (2) allowed isolation of greater numbers of high-producing clones, (3) resulted in greater mRNA recovery per recombinant gene copy, (4) retained stable mRNA and protein expression after amplification provided Methotrexate (MTX) was present (but not in the absence of MTX when instability was observed) and (5) conferred copy number-dependent expression to linked transgene, suggesting that they are resistant to positional gene-silencing effects. It was concluded that the inclusion of UCOEs within expression constructs offers significant advantages for certainty of cell line generation (and the number of recovered clones for more detailed characterisation/optimisation) and that UCOEs are compatible with DHFR amplification protocols. The data suggested that enhanced cell line recovery by transcriptional enhancement of selection markers, such as DHFR, could be achieved.

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry. In the creation of mammalian cell lines plasmid DNA carrying the gene-of-interest integrates randomly into the host cell genome, which results in variable levels of gene expression between cell lines due to gene silencing mechanisms. In addition, cell lines often show unstable protein production during long-term culture. This means that a large number of clones need to be screened in order to isolate stable, high producing cell lines making mammalian cell line development a long and laborious process. In this study an expression platform incorporating a Ubiquitous Chromatin Opening Element (UCOE; which are proposed to maintain chromatin in an open state) has been utilised for the expression of eGFP in CHO cells. Cell lines containing a UCOE vector, showed a significantly higher and more consistent eGFP expression than the non-UCOE cell lines without DHFR amplification. To further improve recombinant protein production cell lines were amplified with methotrexate (MTX). UCOE cell lines showed improved growth in MTX therefore amplification to 250 nM MTX was achieved following a one-step amplification procedure. However, non-UCOE cell lines showed higher levels of eGFP production following MTX amplification. In addition, UCOE cell lines did not improve stability during long-term culture in the absence of selective pressure. Stable eGFP production was achieved for all cell lines when MTX is present. Finally, UCOE cell lines displayed more consistent response to external stimuli than non-UCOE cell lines, suggesting that UCOE cell lines are less prone to clonal variability.

Culture systems based on spin tube reactors have been consolidated in the development of manufacturing processes based on Chinese hamster ovary (CHO) cells.Despite their widespread use, there is little information about the consequences of varying operational setting parameters on the culture performance of recombinant CHO cell lines. Here, we investigated the effect of varying working volumes and agitation speeds on cell growth, protein production, and cell metabolism of two clonally

The requirement for complex therapeutic proteins has resulted in mammalian cells, especially CHO cells, being the dominant host for recombinant protein manufacturing. In creating recombinant CHO cell lines, the expression vectors integrate into various parts of the genome leading to variable levels of expression and stability of protein production. This makes mammalian cell line development a long and laborious process. Therefore, with the intention to accelerate process development of recombinant protein production in CHO systems, UCOEs are utilized to diminish instability of production by maintaining an open chromatin surrounding in combination with MTX amplification. Chromosome painting and FISH analysis were performed to provide detailed molecular evaluation on the location of amplified genes and its relationship to the productivity and stability of the amplified cell lines. In summary, cell lines generated with vectors containing UCOEs retained stable GFP expression with MTX present (but instability was observed in the absence of MTX). UCOE cell lines displayed a higher frequency of integration into>1 chromosome than non-UCOE group. Cell populations were more homogenous in terms of transgene location at the end of Long-term culture (LTC). Overall our findings suggest variation in eGFP fluorescence may be attributed to changes in transgene integration profile over LTC.