HighlightsTransient overexpression of miR-17 and miR-17–92 cluster enhanced growth rate.Biological effects of long term and stable overexpression of miRNAs in batch cultures were studied.Stable miR-17 engineered CHO cells had both improved growth rate and productivity.

Chinese hamster ovary (CHO) cells are the predominant cell factory for the production of recombinant therapeutic proteins. Nevertheless, the lack in publicly available sequence information is severely limiting advances in CHO cell biology, including the exploration of microRNAs (miRNA) as tools for CHO cell characterization and engineering. In an effort to identify and annotate both conserved and novel CHO miRNAs in the absence of a Chinese hamster genome, we deep-sequenced small RNA fractions of 6 biotechnologically relevant cell lines and mapped the resulting reads to an artificial reference sequence consisting of all known miRNA hairpins. Read alignment patterns and read count ratios of 5′ and 3′ mature miRNAs were obtained and used for an independent classification into miR/miR* and 5p/3p miRNA pairs and discrimination of miRNAs from other non-coding RNAs, resulting in the annotation of 387 mature CHO miRNAs. The quantitative content of next-generation sequencing data was analyzed and confirmed using qPCR, to find that miRNAs are markers of cell status. Finally, cDNA sequencing of 26 validated targets of miR-17-92 suggests conserved functions for miRNAs in CHO cells, which together with the now publicly available sequence information sets the stage for developing novel RNAi tools for CHO cell engineering.

During the process of recombinant cell line optimisation for production of biopharmaceuticals, multiple cellular properties like robustness against stress, the attainment of high cell concentrations and maintenance of high viability must be considered to maximize protein yield. To improve growth and viability, glutamine is supplemented as an alternative energy source for rapidly dividing cells that oxidize glucose inefficiently. However, the resulting by‐product ammonia is toxic at high concentrations and has a negative impact on protein glycosylation, a major quality‐determining parameter of biopharmaceuticals. In this work, the CHO‐K1 cell line was adapted to a chemically defined medium and suspension growth within 3 weeks. Subsequently, the glutamine concentration was stepwise reduced from 8 to 4 and 2 mM. After each reduction, both the final cell concentration in the batch and the viability decreased. To force a rapid evolution of cells to achieve high final cell concentrations, cells were seeded at high densities (107 cells/mL) and surviving cells were sorted by FACS or MACS when viability declined to 10% (typically after 24 h). Sorted cells were grown in batch until viability declined to 10% and viable cells recovered again. The final sorted population was able to reach comparable or even better viable cell concentrations and showed a significantly improved viability compared to their ancestors. The 2 mM glutamine‐adapted cell line was directly transferred into glutamine‐free medium and was able to grow at comparable rates without requiring further adaptation. Cells compensated the lack of glutamine by increasing their consumption of glutamate and aspartate.

In spite of the importance of Chinese hamster ovary (CHO) cells for recombinant protein production, very little is known about the molecular and gene regulatory mechanisms that control cellular phenotypes such as enhanced growth under serum-free conditions or high productivity. Most microarray analyses to this purpose are performed with samples taken during the exponential growth phase. However, the cellular transcriptome is dynamic, changing in response to external and internal stimuli and thus reflecting the current functional capacity of cells as well as their ability to adapt to a changing environment. Therefore, during batch or fed-batch cultivations it can be expected that the transcription pattern of genes will change and that such changes may give indications on the cellular state in terms of viability, growth, and productivity. In the current study we monitored the change in expression patterns of mRNAs and microRNAs (miRNA) during lag, exponential, and stationary phases in CHO-K1 suspension cell cultures. In total, over 1400 mRNAs and more than 100 miRNAs were differentially regulated (p<0.05) relative to the batch culture at the starting point. Functional clustering revealed groups of genes with similar expression patterns, which were subjected to functional pathway analysis. In addition, as miRNAs generally act as negative post-transcriptional regulators of mRNAs, we looked for changes in their expression that were inverse to those of their predicted target mRNAs.