Stable microRNA expression enhances therapeutic antibody productivity of Chinese hamster ovary cells

Abstract

MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally regulate the expression of different target genes and, thus, enable engineered gene networks to achieve complex phenotypic changes in mammalian cells. We hypothesized that exploiting this feature of miRNAs could improve therapeutic protein production processes by increasing viable cell densities and/or productivity of the mammalian cells used for manufacturing. To identify miRNAs that increase the productivity of producer cells, we performed a genome wide functional miRNA screen by transient transfection of Chinese hamster ovary (CHO) cells stably expressing an IgG1 antibody (CHO-IgG1). Using this approach, we identified nine human miRNAs that improved the productivities not only of the CHO-IgG1 cells but also of CHO cells expressing recombinant human serum albumin (HSA), demonstrating that the miRNAs act in a product-independent manner. We selected two miRNAs (miR-557 and miR-1287) positively impacting the viable cell density and the specific productivity, respectively, and then stably co-expressed them in IgG1 expressing CHO cells. In these cells, higher IgG1 titers were observed in fed-batch cultures whilst product quality was conserved, demonstrating that miRNA-based cell line engineering provides an attractive approach toward the genetic optimization of CHO producer cells for industrial applications.

MiRNA mimic screen for improved expression of functional neurotensin receptor from HEK 293 cells

“…This approach has previously proven effective for apoptosis screening (Druz et al 2013a) and recombinant secreted protein screening (Fischer et al 2014; Strotbek et al 2013)in CHO cells. In this study, we developed an image-based high-throughput screening method to detect per cell green fluorescence signal, which is applied as a proxy for the number of molecules of NTSR1 protein expressed per cell.…”

3.1 Cell Line Evolution and Engineering

“…Moreover, a recent functional miRNA screen in IgG producing CHO cells identified several miRNAs that enhanced productivity upon transient expression. Stable coexpression of 2 of those miRNAs lead to higher IgG titers in fed-batch cultures [49], suggesting that miRNA engineering may be an encouraging approach to fundamentally reprogram production cells.…”

“…However, it still takes thorough studies to investigate each one of those hypotheses by generating and assessing novel phenotypes. In the future it will be particularly important to directly screen for novel phenotypes in cell culture environments that resemble the actual bioprocesses as closely as possible [49]. Miniaturized automated bioreactor systems that allow running multiple fed-batch cultures in parallel are one approach that seems useful to screen broadly and in relevant systems.…”

Identification of microRNAs specific for high producer CHO cell lines using steady-state cultivation

“…In mammalian cells, miRNAs are predicted to regulate or fine-tune gene expression of ~50 % of all protein-coding genes (Krol et al 2010). In CHO cells, it has already been shown that miRNAs can be utilized to improve growth (Jadhav et al 2012), apoptosis resistance (Druz et al 2013), and specific productivity (Barron et al 2011; Jadhav et al 2014; Strotbek et al 2013). …”