Nature as blueprint: Global phenotype engineering of CHO production cells based on a multi-omics comparison with plasma cells.

Especially for the production of artificial, difficult to express molecules a further development of the CHO production cell line is required to keep pace with the continuously increasing demands. However, the identification of novel targets for cell line engineering to improve CHO cells is a time and cost intensive process. Since plasma cells are evolutionary optimized for a high antibody expression in mammals, we performed a comprehensive multi-omics comparison between CHO and plasma cells to exploit optimized cellular production traits. Comparing the transcriptome, proteome, miRNome, surfaceome and secretome of both cell lines identified key differences including 392 potential overexpression targets for CHO cell engineering categorized in 15 functional classes like transcription factors, protein processing or secretory pathway. In addition, 3 protein classes including 209 potential knock-down/out targets for CHO engineering were determined likely to affect aggregation or proteolysis. For production phenotype engineering, several of these novel targets were successfully applied to transient and transposase mediated overexpression or knock-down strategies to efficiently improve productivity of CHO cells. Thus, substantial improvement of CHO productivity was achieved by taking nature as a blueprint for cell line engineering.

Spatial Proteomics Reveals Differences in the Cellular Architecture of Antibody-Producing CHO and Plasma Cell-Derived Cells.

Most of the recombinant biotherapeutics employed today to combat severe illnesses, for example, various types of cancer or autoimmune diseases, are produced by Chinese hamster ovary (CHO) cells. To meet the growing demand of these pharmaceuticals, CHO cells are under constant development in order to enhance their stability and productivity. The last decades saw a shift from empirical cell line optimization toward rational cell engineering using a growing number of large omics datasets to alter cell physiology on various levels. Especially proteomics workflows reached new levels in proteome coverage and data quality because of advances in high-resolution mass spectrometry instrumentation. One type of workflow concentrates on spatial proteomics by usage of subcellular fractionation of organelles with subsequent shotgun mass spectrometry proteomics and machine learning algorithms to determine the subcellular localization of large portions of the cellular proteome at a certain time point. Here, we present the first subcellular spatial proteome of a CHO-K1 cell line producing high titers of recombinant antibody in comparison to the spatial proteome of an antibody-producing plasma cell-derived myeloma cell line. Both cell lines show colocalization of immunoglobulin G chains with chaperones and proteins associated in protein glycosylation within the endoplasmic reticulum compartment. However, we report differences in the localization of proteins associated to vesicle-mediated transport, transcription, and translation, which may affect antibody production in both cell lines. Furthermore, pairing subcellular localization data with protein expression data revealed elevated protein masses for organelles in the secretory pathway in plasma cell-derived MPC-11 (Merwin plasma cell tumor-11) cells. Our study highlights the potential of subcellular spatial proteomics combined with protein expression as potent workflow to identify characteristics of highly efficient recombinant protein-expressing cell lines. Data are available via ProteomeXchange with identifier PXD029115.

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.

Life at the periphery: what makes CHO cells survival talents.

The production of biopharmaceuticals relies on robust cell systems that can produce recombinant proteins at high levels and grow and survive in the stressful bioprocess environment. Chinese hamster ovary cells (CHO) as the main production hosts offer a variety of advantages including robust growth and survival in a bioprocess environment. Cell surface proteins are of special interest for the understanding of how CHO cells react to their environment while maintaining growth and survival phenotypes, since they enable cellular reactions to external stimuli and potentially initiate signaling pathways. To provide deeper insight into functions of this special cell surface sub-proteome, pathway enrichment analysis of the determined CHO surfaceome was conducted. Enrichment of growth/ survival-pathways such as the phosphoinositide-3-kinase (PI3K)-protein kinase B (AKT), mitogen-activated protein kinase (MAPK), Janus kinase/signal transducers and activators of transcription (JAK-STAT), and RAP1 pathways were observed, offering novel insights into how cell surface receptors and ligand-mediated signaling enable the cells to grow and survive in a bioprocess environment. When supplementing surfaceome data with RNA expression data, several growth/survival receptors were shown to be co-expressed with their respective ligands and thus suggesting self-induction mechanisms, while other receptors or ligands were not detectable. As data about the presence of surface receptors and their associated expressed ligands may serve as base for future studies, further pathway characterization will enable the implementation of optimization strategies to further enhance cellular growth and survival behavior. KEY POINTS: • PI3K/AKT, MAPK, JAK-STAT, and RAP1 pathway receptors are enriched on the CHO cell surface and downstream pathways present on mRNA level. • Detected pathways indicate strong CHO survival and growth phenotypes. • Potential self-induction of surface receptors and respective ligands.

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.

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.

Development of Responsive Promoters and their Utilization for Stable CHO Sensor Cell Lines.

Chinese hamster ovary (CHO) cells are the most important mammalian expression systems to produce recombinant proteins. To ensure a proper expression of the desired molecule, it is important to monitor and adjust bioprocess parameters like oxygen concentration as well as osmolality. However, the observation of crucial cultivation parameters can be an elaborate procedure requiring lots of hands-on work. In addition, for emerging modeling approaches for bioprocesses, a model cell line responding with a measurable signal to an external influence would be highly valuable. This protocol describes in detail the procedure to generate responsive promoters reacting to limiting conditions as well as the generation of stable sensor cell lines communicating with the operator. Thereby, hypoxia and osmolality sensing response elements established in CHO cells will be utilized to trigger the expression of a minimal CMV promoter. To assess the activity of the responsive promoter in close to real time, unstable variants of GFP and BFP will be expressed, which can be analyzed via flow cytometry. Finally, an automated sampling system coupled to a fluorescence microscope enables a continuous observation of CHO cells and reports emerging limiting conditions by detecting increasing amounts of a specific fluorescent protein.

The new frontier in CHO cell line development: From random to targeted transgene integration technologies.

Cell line development represents a crucial step in the development process of a therapeutic glycoprotein. Chinese hamster ovary (CHO) cells are the most frequently employed mammalian host cell system for the industrial manufacturing of biologics. The predominant application of CHO cells for heterologous recombinant protein expression lies in the relative simplicity of stably introducing ectopic DNA into the CHO host cell genome. Since CHO cells were first used as expression host for the industrial production of biologics in the late 1980s, stable genomic transgene integration has been achieved almost exclusively by random integration. Since then, random transgene integration had become the gold standard for generating stable CHO production cell lines due to a lack of viable alternatives. However, it was eventually demonstrated that this approach poses significant challenges on the cell line development process such as an increased risk of inducing cell line instability. In recent years, significant discoveries of new and highly potent (semi)-targeted transgene integration systems have paved the way for a technological revolution in the cell line development sector. These advanced methodologies comprise the application of transposase-, recombinase- or Cas9 nuclease-mediated site-specific genomic integration techniques, which enable a scarless transfer of the transgene expression cassette into transcriptionally active loci within the host cell genome. This review summarizes recent advancements in the field of transgene integration technologies for CHO cell line development and compare them to the established random integration approach. Moreover, advantages and limitations of (semi)-targeted integration techniques are discussed, and benefits and opportunities for the biopharmaceutical industry are outlined.

In pursuit of a minimal CHO genome: Establishment of large-scale genome deletions.

Chinese hamster ovary (CHO) cells are the most commonly used mammalian cell line for the production of complex therapeutic glycoproteins. As CHO cells have evolved as part of a multicellular organism, they harbor many cellular functions irrelevant for their application as production hosts in industrial bioprocesses. Consequently, CHO cells have been the target for numerous genetic engineering efforts in the past, but a tailored host cell chassis holistically optimized for its specific task in a bioreactor is still missing. While the concept of genome reduction has already been successfully applied to bacterial production cells, attempts to create higher eukaryotic production hosts exhibiting reduced genomes have not been reported yet. Here, we present the establishment and application of a large-scale genome deletion strategy for targeted excision of large genomic regions in CHO cells. We demonstrate the feasibility of genome reduction in CHO cells using optimized CRISPR/Cas9 based experimental protocols targeting large non-essential genomic regions with high efficiency. Achieved genome deletions of non-essential genetic regions did not introduce negative effects on bioprocess relevant parameters, although we conducted the largest reported genomic excision of 864 kilobase pairs in CHO cells so far. The concept presented serves as a directive to accelerate the development of a significantly genome-reduced CHO host cell chassis paving the way for a next generation of CHO cell factories.

Exploring synthetic biology for the development of a sensor cell line for automated bioprocess control.

Unfavorable process conditions lead to adverse cultivation states, limited cell growth and thus hamper biotherapeutic protein production. Oxygen deficiency or hyperosmolality are among the most critical process conditions and therefore require continuous monitoring. We established a novel sensor CHO cell line with the ability to automatically sense and report unwanted process conditions by the expression of destabilized fluorescent proteins. To this end, an inducible real-time system to detect hypoxia by hypoxia response elements (HREs) of vascular endothelial growth factor (VEGF) origin reporting limitations by the expression of destabilized green fluorescent protein (GFP) was created. Additionally, we established a technique for observing hyperosmolality by exploiting osmotic response elements (OREs) for the expression of unstable blue fluorescent protein (BFP, FKBP-BFP), enabling the simultaneous automated supervision of two bioprocess parameters by using a dual sensor CHO cell line transfected with a multiplexable monitoring system. We finally also provided a fully automated in-line fluorescence microscopy-based setup to observe CHO cells and their response to varying culture conditions. In summary, we created the first CHO cell line, reporting unfavorable process parameters to the operator, and provided a novel and promising sensor technology accelerating the implementation of the process analytical technology (PAT) initiative by innovative solutions.

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.

Cell line development for continuous high cell density biomanufacturing: Exploiting hypoxia for improved productivity.

Oxygen deficiency (hypoxia) induces adverse effects during biotherapeutic protein production leading to reduced productivity and cell growth. Hypoxic conditions occur during classical batch fermentations using high cell densities or perfusion processes. Here we present an effort to create novel engineered Chinese hamster ovary (CHO) cell lines by exploiting encountered hypoxic bioprocess conditions to reinforce cellular production capacities. After verifying the conservation of the hypoxia-responsive pathway in CHO cell lines by analyzing oxygen sensing proteins HIF1a, HIF1ß and VDL, hypoxia-response-elements (HREs) were functionally analyzed and used to create hypoxia-responsive expression vectors. Subsequently engineered hypoxia sensitive CHO cell lines significantly induced protein expression (SEAP) during adverse oxygen limitation encountered during batch fermentations as well as high cell density perfusion processes (2.7 fold). We also exploited this novel cell system to establish a highly effective oxygen shift as innovative bioprocessing strategy using hypoxia induction to improve production titers. Thus, substantial improvements can be made to optimize CHO cell productivity for novel bioprocessing challenges as oxygen limitation, providing an avenue to establish better cell systems by exploiting adverse process conditions for optimized biotherapeutic production.

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.