Loss of a newly discovered microRNA in Chinese hamster ovary cells leads to upregulation of N-glycolylneuraminic acid sialylation on monoclonal antibodies.

Chinese hamster ovary (CHO) cells are known not to express appreciable levels of the sialic acid residue N-glycolylneuraminic acid (NGNA) on monoclonal antibodies. However, we actually have identified a recombinant CHO cell line expressing an IgG with unusually high levels of NGNA sialylation (>30%). Comprehensive multi-OMICs based experimental analyses unraveled the root cause of this atypical sialylation: (1) expression of the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene was spontaneously switched on, (2) CMAH mRNA showed an anti-correlated expression to the newly discovered Cricetulus griseus (cgr) specific microRNA cgr-miR-111 and exhibits two putative miR-111 binding sites, (3) miR-111 expression depends on the transcription of its host gene SDK1, and (4) a single point mutation within the promoter region of the sidekick cell adhesion molecule 1 (SDK1) gene generated a binding site for the transcriptional repressor histone H4 transcription factor HINF-P.

A blueprint from nature: miRNome comparison of plasma cells and CHO cells to optimize therapeutic antibody production.

Chinese Hamster Ovary (CHO) cells are the most frequently used biopharmaceutical production hosts, although industry is presently suffering from their variable recombinant product quality, insufficient long-term stability and low productivity. Here, we present an effort to address overall cell line engineering by a novel bottom-up microRNA (miRNA) screening approach. miRNAs are small non-coding RNAs known to regulate global gene expression at the post-transcriptional level and have proved to serve as promising tools for cell line engineering for over a decade.

Unveiling the CHO surfaceome: Identification of cell surface proteins reveals cell aggregation-relevant mechanisms.

Chinese hamster ovary (CHO) suspension cells are the main production hosts for biopharmaceuticals. For the improvement of production processes, it is essential to understand the interaction between CHO cells and their microenvironment. While the cellular membrane is the crucial surface barrier between the inner and outer cell compartments, the subgroup of cell surface proteins (surfaceome) is of particular interest due to its potential to react to external factors and initiate cell communication and interaction pathways.

Rational optimization of a monoclonal antibody improves the aggregation propensity and enhances the CMC properties along the entire pharmaceutical process chain.

The discovery of therapeutic monoclonal antibodies (mAbs) primarily focuses on their biological activity favoring the selection of highly potent drug candidates. These candidates, however, may have physical or chemical attributes that lead to unfavorable chemistry, manufacturing, and control (CMC) properties, such as low product titers, conformational and colloidal instabilities, or poor solubility, which can hamper or even prevent development and manufacturing. Hence, there is an urgent need to consider the developability of mAb candidates during lead identification and optimization.

Unraveling what makes a monoclonal antibody difficult-to-express: From intracellular accumulation to incomplete folding and degradation via ERAD.

Although most therapeutic monoclonal antibodies (mAbs) can routinely be produced in the multigram per litre range, some mAb candidates turn out to be difficult-to-express (DTE). In addition, the class of more complex biological formats is permanently increasing and mammalian expression systems like Chinese hamster ovary (CHO) cell lines can show low performance. Hence, there is an urgent need to identify any rate limiting processing step during cellular synthesis.

Human CAP cells represent a novel source for functional, miRNA-loaded exosome production.

Exosomes represent a promising delivery tool for nucleic acid-based pharmaceuticals. They are highly suitable for transporting therapeutic miRNAs to tumor cells, due to their natural membrane components. Further, exosomes are capable of effectively protecting nucleic acids against ribonucleases and enable the delivery of their content through cell membranes.

CRISPR/Cas9-Mediated Knockout of MicroRNA-744 Improves Antibody Titer of CHO Production Cell Lines.

MicroRNAs (miRNAs) are noncoding RNAs that serve as versatile molecular engineering tools to improve production cells by overexpression or knockdown of miRNAs showing beneficial or adverse effects on cell-culture performance. The genomic knockout (KO) of noncoding RNAs in Chinese hamster ovary (CHO) production cells has not been reported. However, given the significant number of miRNAs showing negative effects on CHO-bioprocess performance and the development of clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas9), genome editing tools facilitate precise optimization of CHO cells via modulation of noncoding RNAs.

Visualisation of intracellular production bottlenecks in suspension-adapted CHO cells producing complex biopharmaceuticals using fluorescence microscopy.

With the advance of complex biological formats such as bispecific antibodies or fusion proteins, mammalian expression systems often show low performance. Described determining factors may be accumulation or haltering of heterologous proteins within the different cellular compartments disturbing transport or secretion. In case of the investigated bispecific antibody (bsAb)-producing Chinese hamster ovary (CHO) cell line neither impaired transcription nor decreased translation processes were identified and thus satisfactorily explained its low production capacity.

Enhanced protein production by microRNA-30 family in CHO cells is mediated by the modulation of the ubiquitin pathway.

Functional genomics represent a valuable approach to improve culture performance of Chinese hamster ovary (CHO) cell lines for biopharmaceutical manufacturing. Recent advances in applied microRNA (miRNAs) research suggest that these small non-coding RNAs are critical for the regulation of cell phenotypes in CHO cells. However, the notion that individual miRNAs usually control the expression of hundreds of different genes makes miRNA target identification highly complex.

miR-2861 as novel HDAC5 inhibitor in CHO cells enhances productivity while maintaining product quality.

Histone deacetylase (HDAC) inhibitors have been exploited for years to improve recombinant protein expression in mammalian production cells. However, global HDAC inhibition is associated with negative effects on various cellular processes. microRNAs (miRNAs) have been shown to regulate gene expression in almost all eukaryotic cell types by controlling entire cellular pathways.

A functional high-content miRNA screen identifies miR-30 family to boost recombinant protein production in CHO cells.

The steady improvement of mammalian cell factories for the production of biopharmaceuticals is a key challenge for the biotechnology community. Recently, small regulatory microRNAs (miRNAs) were identified as novel targets for optimizing Chinese hamster ovary (CHO) production cells as they do not add any translational burden to the cell while being capable of regulating entire physiological pathways. The aim of the present study was to elucidate miRNA function in a recombinant CHO-SEAP cell line by means of a genome-wide high-content miRNA screen.