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. Here the miRNome of plasma cells (PCs) has been analyzed as the natural blueprint for optimized production and secretion of antibodies. Performing comparative miRNome cross-species expression analysis of four murine/human PC-derived (PCD) and two CHO cell lines showed 147 conserved miRNAs to be differentially expressed between PCDs and CHOs. Conducting a targeted miRNA screen of this PC-specific miRNA subset revealed 14 miRNAs to improve bioprocess relevant parameters in CHO cells, among them the PC-characteristic miR-183 cluster. Finally, miRNA target prediction tools and transcriptome analysis were combined to elucidate differentially regulated lysine degradation and fatty acid metabolism pathways in monoclonal antibody (mAb) expressing CHO-DG44 and CHO-K1 cells, respectively. Thus, substantial new insights into molecular and cellular mechanisms of biopharmaceutical production cell lines can be gained by targeted bottom-up miRNA screenings.

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. The resulting transcriptional repression of SDK1 led to a downregulation of its co-expressed miR-111 and hence to a spontaneous upregulation of CMAH expression finally increasing NGNA protein sialylation.

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. Therefore, the CHO surfaceome was explored for the first time by enriching exposed N-glycosylated membrane proteins before tandem mass spectrometry (MS/MS) analyses, identifying a total of 449 surface proteins, including 34 proteins specific for production cells. Functional annotation and classification located most proteins to the cell surface belonging mainly to the protein classes of receptors, enzymes, and transporters. In addition, adhesion molecules as cadherins, integrins, Ig superfamily and extracellular matrix (ECM) proteins as collagens, laminins, thrombospondin, fibronectin, and tenascin were significantly enriched, which are involved in mechanisms for the formation of cell junctions, cell-cell and cell-ECM adhesion as focal adhesions. As cell adhesion and aggregation counteracts scalable production of biopharmaceuticals, experimental validation confirmed differential expression of integrin ß1 (ITGB1) and ß3, CD44, laminin, and fibronectin on the surface of aggregation-prone CHO production cells. The subsequent modulation of the central interaction protein ITGB1 by small interfering RNA knockdown substantially counteracted cell aggregation pointing toward novel engineering routes for aggregation reduction in biopharmaceutical production cells and exemplifying the potential of the surfaceome for specified engineering strategies.

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. This work provides a comprehensive proof of concept study for the significantly improved developability of a mAb variant that was optimized with the help of sophisticated in silico tools relative to its difficult-to-develop parental counterpart. Interestingly, a single amino acid substitution in the variable domain of the light chain resulted in a three-fold increased product titer after stable expression in Chinese hamster ovary cells. Microscopic investigations revealed that wild type mAb-producing cells displayed potential antibody inclusions, while the in silico optimized variant-producing cells showed a rescued phenotype. Notably, the drug substance of the in silico optimized variant contained substantially reduced levels of aggregates and fragments after downstream process purification. Finally, formulation studies unraveled a significantly enhanced colloidal stability of the in silico optimized variant while its folding stability and potency were maintained. This study emphasizes that implementation of bioinformatics early in lead generation and optimization of biotherapeutics reduces failures during subsequent development activities and supports the reduction of project timelines and resources.

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. Therefore, we assessed the intracellular location of the DTE antibody mAb2 by fluorescence and electron microscopy (EM) and revealed an accumulation of the antibody, which led to an aberrant morphology of the endoplasmic reticulum (ER). Analysis of underlying cellular mechanisms revealed that neither aggregation nor antibody assembly, but folding represented the reason for hampered secretion. We identified that the disulfide bridge formation within the antibody light chain (LC) was impaired due to less recognition by protein disulfide isomerase (PDI). As a consequence, the DTE molecule was degraded intracellularly by the ubiquitin proteasome system via ER-associated degradation (ERAD). This study revealed that with the continuous emergence of DTE therapeutic protein candidates, special attention needs to be drawn to optimization processes to ensure manufacturability.

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. However, no suitable production host for miRNA containing exosomes of non-tumorigenic origin has yet been identified. In this study we engineered an immortalised human amniocyte cell line (CAP® cells), whose exosomes were enriched and characterised. The cell line modifications not only enabled the production of GFP-labelled but also pro-apoptotic miRNA containing exosomes without negative influence on host cell growth. Furthermore, we demonstrated that pro-apoptotic miRNA containing CAP exosomes are taken up by ovarian cancer cells. Strikingly, delivery of functional exosomal miRNA led to downregulation of several reported target genes in the treated tumor cells. In summary, we revealed CAP cells of non-tumorigenic origin as a novel and efficient exosome production host with the potential to produce functional miRNA-loaded exosomes.

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. In a previous high-content miRNA screen, miR-744 was identified as a potential target associated with reduced productivity. Hence, the genomic miR-744 precursor sequence is deleted by two single guide RNA (sgRNA)-Cas9-mediated DNA double-strand breaks (DSB) flanking the miR-744 locus. After fluorescence-activated cell sorting (FACS), clonal miR-744 KO cell lines are recovered and three of them are confirmed as miR-744 KOs. Impacts of CRISPR/Cas9 editing are characterized at the genetic, transcript, and phenotypic levels. During batch cultivation, antibody titers of miR-744 KOs are significantly increased to 190-311 mg L-1 compared to a nontargeting (NT) sgRNA transfected clonal control with 156 mg L-1 , pointing towards the potential of miRNA KO for cell line engineering.

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. Hence, we established a streamlined confocal microscopy-based methodology for CHO production cells investigating the distribution of the recombinant protein within the respective organelles of the secretory pathway and visualised the structure of the endoplasmic reticulum (ER) to be affected pinpointing towards an intra-ER bottleneck putatively hampering or limiting efficient secretion. The ER displayed not only a heavily altered morphology in comparison to a high immunoglobulin G (IgG)-producing cell line with a possibly inflated or overloaded structure, but the recombinant protein was also completely absent in the Golgi apparatus. Notably, the results obtained using an automated microscopy approach suggest the possible application of this methodology in cell line development and engineering.

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. We have recently reported that the entire miR-30 family enhances recombinant protein production in CHO cells. To better understand the pro-productive effects of this miRNA family, we set out to identify their downstream target genes in CHO cells. Computational target prediction combined with a comprehensive functional validation enabled the discovery of a set of twenty putative target genes for all productivity enhancing miR-30 family members. We demonstrate that all miR-30 isoforms contribute to the regulation of the ubiquitin pathway in CHO cells by directly targeting the ubiquitin E3 ligase S-phase kinase-associated protein 2 (Skp2). Finally, we provide several lines of evidence that miR-30-mediated modulation of the ubiquitin pathway may enhance recombinant protein expression in CHO cells. In summary, this study supports the importance of non-coding RNAs, especially of miRNAs, in the context of cell line engineering.

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. Since miRNAs recently have gained much attention as next-generation cell engineering tool to improve Chinese hamster ovary (CHO) cell factories, we were interested if miRNAs are able to specifically repress HDAC expression in CHO cells to circumvent limitations of unspecific HDAC inhibition. We discovered a novel miRNA in CHO cells, miR-2861, which was shown to enhance productivity in various recombinant CHO cell lines. Furthermore, we demonstrate that miR-2861 might post-transcriptionally regulate HDAC5 in CHO cells. Intriguingly, siRNA-mediated HDAC5 suppression could be demonstrated to phenocopy pro-productive effects of miR-2861 in CHO cells. This supports the notion that miRNA-induced inhibition of HDAC5 may contribute to productivity enhancing effects of miR-2861. Furthermore, since product quality is fundamental to safety and functionality of biologics, we examined the effect of HDAC inhibition on critical product quality attributes. In contrast to unspecific HDAC inhibition using VPA, enforced expression of miR-2861 did not negatively influence antibody aggregation or N-glycosylation. Our findings highlight the superiority of miRNA-mediated inhibition of specific HDACs and present miR-2861 as novel cell engineering tool for improving CHO manufacturing cells.

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. This screen revealed that out of the 1, 139 miRNAs examined, 21% of the miRNAs enhanced cell-specific SEAP productivity mainly resulting in elevated volumetric yields, while cell proliferation was accelerated by 5% of the miRNAs. Conversely, cell death was diminished by 13% (apoptosis) or 4% (necrosis) of all transfected miRNAs. Besides these large number of identified target miRNAs, the outcome of our studies suggest that the entire miR-30 family substantially improves bioprocess performance of CHO cells. Stable miR-30 over expressing cells outperformed parental cells by increasing SEAP productivity or maximum cell density of approximately twofold. Our results highlight the application of miRNAs as powerful tools for CHO cell engineering, identified the miR-30 family as a critical component of cell proliferation, and support the notion that miRNAs are powerful determinants of cell viability.