miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495-1510. © 2017 Wiley Periodicals, Inc.

ATF6ß-based fine-tuning of the unfolded protein response enhances therapeutic antibody productivity of Chinese hamster ovary cells.

The dynamics of protein folding and secretion are key issues in improving the productivity and robustness of Chinese hamster ovary (CHO) producer cells. High recombinant protein secretion in CHO producer clones triggers the activation of the unfolded protein response (UPR), an intracellular response to the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER). We previously reported that the human microRNA (miRNA) miR-1287 enhances productivity in IgG-expressing CHO cells (CHO-IgG). Here, through next-generation sequencing (NGS), we identified the activating transcription factor 6 beta (ATF6ß), a repressor of the pro-survival and UPR promoting factor ATF6α, as a direct target gene of miR-1287 in CHO-IgG cells. We show that the transient depletion of ATF6ß resulted in enhanced specific productivity comparable to that of miR-1287-expressing CHO-IgG cells. Strikingly, stable ATF6ß knockdown in CHO-IgG cells significantly improved antibody titer and viable cell density under fed-batch conditions. This was associated with the elevated expression of the UPR genes glucose-regulated protein 78 (GRP78), homocysteine inducible ER protein with ubiquitin like domain 1 (Herpud1) and CCAAT/enhancer-binding protein homologous protein (CHOP). We hence demonstrate that ATF6ß-based cell line engineering is a promising strategy to improve the productivity of CHO producer cells by activating an optimally balanced UPR program. Biotechnol. Bioeng. 2017;114: 1310-1318. © 2017 Wiley Periodicals, Inc.

Secretory pathway optimization of CHO producer cells by co-engineering of the mitosRNA-1978 target genes CerS2 and Tbc1D20.

Chinese Hamster Ovary (CHO) cells are the most commonly used host for the production of biopharmaceuticals. Although transcription and translation engineering strategies have been employed to generate high-producer cell clones, the secretory pathway still remains a bottleneck in cellular productivity. In this study we show that ectopic expression of a human mitochondrial genome-encoded small RNA (mitosRNA-1978) in an IgG expressing CHO cell line strongly improved specific productivity by functioning in a microRNA-like fashion. By next generation sequencing we identified two endoplasmic reticulum (ER)-localized proteins, Ceramide Synthase 2 (CerS2) and the Rab1 GAP Tbc domain family member 20 (Tbc1D20), as target genes of mitosRNA-1978. Combined transient siRNA-mediated knockdown of CerS2 and Tbc1D20 resulted in increased specific productivity of CHO-IgG cells, thus recapitulating the mitosRNA-1978 phenotype. In support of a function in vesicular trafficking at the level of the ER, we provide evidence for altered cellular ceramide composition upon CerS2 knockdown and increased activity of Rab1 in CHO-IgG cells depleted of Tbc1D20. Importantly, in a fed-batch process, the combined stable knockdown of CerS2 and Tbc1D20 in CHO-IgG cells resulted in dramatically increased antibody production which was accompanied by enhanced cell growth. Thus, by identifying mitosRNA-1978 target genes in combination with an informed shRNA-mediated co-engineering approach we successfully optimized the secretory capacity of CHO producer cells used for the manufacturing of therapeutic proteins.

To clone or not to clone? Induced pluripotent stem cells can be generated in bulk culture.

Induced pluripotent stem cells (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). Given that fully reprogrammed cells are highly proliferative and escape from cellular senescence, it is conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency was higher and size was larger when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs revealed similar in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs - this is of relevance for standardization and automation of cell culture procedures.