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.

Developing microRNAs as engineering tools to modulate monoclonal antibody galactosylation.

N-linked glycosylation is one of the most important post-translational modifications of monoclonal antibodies (mAbs) and is considered to be a critical quality attribute (CQA), as the glycan composition often has immunomodulatory effects. Since terminal galactose residues of mAbs can affect antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytolysis (CDC) activation, serum half-life, and antiviral activity it has to be monitored, controlled and modulated to ensure therapeutic effects. The ability of small noncoding microRNAs (miRNAs) to modulate glycosylation in Chinese hamster ovary (CHO) production cells was recently reported establishing miRNAs as engineering tools for modulation of protein glycosylation. In this study, we report the characterization and validation of miRNAs as engineering tools for increased (mmu-miR-452-5p, mmu-miR-193b-3p) or decreased (mmu-miR-7646-5p, mmu-miR-7243-3p, mmu-miR-1668, mmu-let-7c-1-3p, mmu-miR-7665-3p, mmu-miR-6403) degree of galactosylation. Furthermore, the biological mode of action regulating gene expression of the galactosylation pathway was characterized as well as their influence on bioprocess-related parameters. Most important, stable plasmid-based overexpression of these miRNAs represents a versatile tool for engineering N-linked galactosylation to achieve favorable phenotypes in cell lines for biopharmaceutical production.

Hyperthermic shift and cell engineering increase small extracellular vesicle production in HEK293F cells.

Although small extracellular vesicles (sEVs) have promising features as an emerging therapeutic format for a broad spectrum of applications, for example, blood-brain-barrier permeability, low immunogenicity, and targeted delivery, economic manufacturability will be a crucial factor for the therapeutic applicability of sEVs. In the past, bioprocess optimization and cell line engineering improved titers of classical biologics multifold. We therefore performed a design of experiments (DoE) screening to identify beneficial bioprocess conditions for sEV production in HEK293F suspension cells. Short-term hyperthermia at 40°C elevated volumetric productivity 5.4-fold while sEVs displayed improved exosomal characteristics and cells retained >90% viability. Investigating the effects of hyperthermia via transcriptomics and proteomics analyses, an expectable, cellular heat-shock response was found together with an upregulation of many exosome biogenesis and vesicle trafficking related molecules, which could cause the productivity boost in tandem with heat shock proteins (HSPs), like HSP90 and HSC70. Because of these findings, a selection of 44 genes associated with exosome biogenesis, vesicle secretion machinery, or heat-shock response was screened for their influence on sEV production. Overexpression of six genes, CHMP1A, CHMP3, CHMP5, VPS28, CD82, and EZR, significantly increased both sEV secretion and titer, making them suitable targets 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.

A novel system for glycosylation engineering by natural and artificial miRNAs.

N-linked glycosylation is a crucial post-translational modification of many biopharmaceuticals, including monoclonal antibodies (mAbs), capable of modifying their biological effect in patients and thus considered as a critical quality attribute (CQA). However, expression of desired and consistent glycosylation patterns remains a constant challenge for the biopharmaceutical industry and constitutes the need for tools to engineer glycosylation. Small non-coding microRNAs (miRNAs) are known regulators of entire gene networks and have therefore the potential of being used as tools for modulation of glycosylation pathways and for glycoengineering. Here, we demonstrate that novel identified natural miRNAs are capable of altering N-linked glycosylation patterns on mAbs expressed in Chinese hamster ovary (CHO) cells. We established a workflow for a functional high-throughput screening of a complete miRNA mimic library and identified 82 miRNA sequences affecting various moieties including galactosylation, sialylation, and α-1,6 linked core-fucosylation, an important glycan feature influencing antibody-dependent cytotoxicity (ADCC). Subsequent validation shed light on the intra-cellular mode of action and the impact on the cellular fucosylation pathway of miRNAs reducing core-fucosylation. While multiplex approaches increased phenotypic effects on the glycan structure, a synthetic biology approach utilizing rational design of artificial miRNAs further enhanced the potential of miRNAs as novel, versatile and tune-able tools for engineering of N-linked glycosylation pathways and expressed glycosylation patterns towards favourable phenotypes.

Production cell analysis and compound-based boosting of small extracellular vesicle secretion using a generic and scalable production platform.

Extracellular vesicles (EVs) are a novel format of advanced therapeutical medicinal products (ATMPs). They can act regenerative or immune-modulatory as cell therapy substitutes or as a platform for designer exosomes. The biotechnological production of therapeutic EVs is still very much uncharted territory so standardized host cells, production setups, and isolation methods are not yet implemented. In this work, we present a tangential flow filtration (TFF) and fast-performance liquid chromatography (FPLC)-based size exclusion chromatography (SEC) purification setup that is compatible for industry applications. Moreover, we evaluated a series of potential host cell lines regarding their EV productivity, characteristics, and biological functionality. It was found that telomerase-immortalized Wharton's jelly mesenchymal stromal cells (WJ-MSC/TERT273) secrete high amounts of EVs per cell with regenerative capabilities. On the other hand, Cevec's amniocyte producer cells® (CAP®) and human embryonic kidney (HEK293) suspension cells are suitable platforms for designer EVs with high yields. Finally, we aimed to boost the EV secretion of HEK293 cells via chemical adjuvants and verified four compounds that heighten cellular EV secretion in a presumably cAMP-dependent manner. A combination of fenoterol, iodoacetamide, and dinitrophenol increased the EV yield in HEK293 cells threefold and cellular secretion rate fivefold.

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.

High-throughput glycosylation analysis of intact monoclonal antibodies by mass spectrometry coupled with capillary electrophoresis and liquid chromatography.

The analysis of monoclonal antibodies glycosylation is a crucial quality control attribute of biopharmaceutical drugs. High throughput screening approaches for antibody glycoform analysis are required in various stages of process optimization. Here, we present high throughput screening suitable mass spectrometry-based workflows for the analysis of intact antibody glycosylation out of cell supernatants. Capillary electrophoresis and liquid chromatography were coupled with quadrupole time-of-flight mass spectrometry or Orbitrap mass spectrometry. Both separation methods offer fast separation (10-15 min) and the capability to prevent the separated cell supernatant matrix to enter the mass spectrometry by post-separation valving. Both mass spectrometry instruments provide comparable results and both are sufficient to determine the glycosylation pattern of the five major glycoforms of the measured antibodies. However, the Orbitrap yields higher sensitivity of 25 µg/mL (CE-nanoCEasy-Orbitrap mass spectrometry) and 5 µg/mL (liquid chromatography-Orbitrap mass spectrometry). Data processing was optimized for a faster processing and easier detection of low abundant glycoforms based on averaged charge-deconvoluted mass spectra. This approach combines a non-target glycoform analysis while yielding the same glycosylation pattern as the traditional approach based on extracted ion traces. The presented methods enable the high throughput screening of the glycosylation pattern of antibodies down to low µg/mL-range out of cell supernatant without any sample preparation.

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.

Exploring the molecular content of CHO exosomes during bioprocessing.

In biopharmaceutical production, Chinese hamster ovary (CHO) cells derived from Cricetulus griseus remain the most commonly used host cell for recombinant protein production, especially antibodies. Over the last decade, in-depth multi-omics characterization of these CHO cells provided data for extensive cell line engineering and corresponding increases in productivity. However, exosomes, extracellular vesicles containing proteins and nucleic acids, are barely researched at all in CHO cells. Exosomes have been proven to be a ubiquitous mediator of intercellular communication and are proposed as new biopharmaceutical format for drug delivery, indicator reflecting host cell condition and anti-apoptotic factor in spent media. Here we provide a brief overview of different separation techniques and subsequently perform a proteome and regulatory, non-coding RNA analysis of exosomes, derived from lab-scale bioreactor cultivations of a CHO-K1 cell line, to lay out reference data for further research in the field. Applying bottom-up orbitrap shotgun proteomics and next-generation small RNA sequencing, we detected 1395 proteins, 144 micro RNA (miRNA), and 914 PIWI-interacting RNA (piRNA) species differentially across the phases of a batch cultivation process. The exosomal proteome and RNA data are compared with other extracellular fractions and cell lysate, yielding several significantly exosome-enriched species. Graphical Abstract KEY POINTS: • First-time comprehensive protein and miRNA characterization of CHO exosomes. • Isolation protocol and time point of bioprocess strongly affect quality of extracellular vesicles. • CHO-derived exosomes also contain numerous piRNA species of yet unknown function.

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.

Induction of apoptosis in ovarian cancer cells by miR-493-3p directly targeting AKT2, STK38L, HMGA2, ETS1 and E2F5.

Apoptosis is a form of directed programmed cell death with a tightly regulated signalling cascade for the destruction of single cells. MicroRNAs (miRNAs) play an important role as fine tuners in the regulation of apoptotic processes. MiR-493-3p mimic transfection leads to the induction of apoptosis causing the breakdown of mitochondrial membrane potential and the activation of Caspases resulting in the fragmentation of DNA in several ovarian carcinoma cell lines. Ovarian cancer shows with its pronounced heterogeneity a very high death-to-incidence ratio. A target gene analysis for miR-493-3p was performed for the investigation of underlying molecular mechanisms involved in apoptosis signalling pathways. Elevated miR-493-3p levels downregulated the mRNA and protein expression levels of Serine/Threonine Kinase 38 Like (STK38L), High Mobility Group AT-Hook 2 (HMGA2) and AKT Serine/Threonine Kinase 2 (AKT2) by direct binding as demonstrated by luciferase reporter assays. Notably, the protein expression of RAF1 Proto-Oncogene, Serine/Threonine Kinase (RAF1) was almost completely downregulated by miR-493-3p. This interaction, however, was indirect and regulated by STK38L phosphorylation. In addition, RAF1 transcription was diminished as a result of reduced transcription of ETS proto-oncogene 1 (ETS1), another direct target of miR-493-3p. Taken together, our observations have uncovered the apoptosis inducing potential of miR-493-3p through its regulation of multiple target genes participating in the extrinsic and intrinsic apoptosis pathway.

Stable miRNA overexpression in human CAP cells: Engineering alternative production systems for advanced manufacturing of biologics using miR-136 and miR-3074.

Chinese hamster ovary (CHO) cells still represent the major production host for therapeutic proteins. However, multiple limitations have been acknowledged leading to the search for alternative expression systems. CEVEC's amniocyte production (CAP) cells are human production cells demonstrated to enable efficient overexpression of recombinant proteins with human glycosylation pattern. However, CAP cells have not yet undergone any engineering approaches to optimize process parameters for a cheaper and more sustainable production of biopharmaceuticals. Thus, we assessed the possibility to enhance CAP cell production capacity via cell engineering using miRNA technology. Based on a previous high-content miRNA screen in CHO-SEAP cells, selected pro-productive miRNAs including, miR-99b-3p, 30a-5p, 329-3p, 483-3p, 370-3p, 219-1-3p, 3074-5p, 136-3p, 30e-5p, 1a-3p, and 484-5p, were shown to act pro-productive and product independent upon transient transfection in CAP and CHO antibody expressing cell lines. Stable expression of miRNAs established seven CAP cell pools with an overexpression of the pro-productive miRNA strand. Subsequent small-scale screening as well as upscaling batch experiments identified miR-136 and miR-3074 to significantly increase final mAb concentration in CAP-mAb cells. Transcriptomic changes analyzed by microarrays identified several lncRNAs as well as growth and apoptosis-related miRNAs to be differentially regulated in CAP-mAb-miR-136 and -miR-3074. This study presents the first engineering approach to optimize the alternative human expression system of CAP-cells.

Temperature-sensitive miR-483 is a conserved regulator of recombinant protein and viral vector production in mammalian cells.

Cell engineering and bioprocess optimizations such as low temperature cultivation represent powerful tools to improve cellular performance and product yields of mammalian production cells. Besides monoclonal antibodies (mABs), novel biotherapeutic formats such as viral vectors will gain increasing importance. Here, we demonstrate that similar to Chinese hamster ovary (CHO) cells, product yields of recombinant adeno-associated virus (rAAV) producing HeLa cells can be markedly increased by low temperature cultivation. MicroRNAs (miRNAs) are small non-coding RNAs that critically regulate cell phenotypes. We thus investigated differential miRNA expression in response to mild hypothermia in CHO and HeLa production cells. We discovered miR-483 to be substantially up-regulated upon temperature down-shift in both cell types. Functional validation experiments revealed that introduction of miR-483 mimics led to a significant increase in both rAAV and mAB production in HeLa and CHO cells, respectively. Furthermore, inhibition of miR-483 up-regulation during mild hypothermia significantly decreased product yields, suggesting that miR-483 is a key regulator of cellular productivity in mammalian cells. In addition, miRNA target gene identification indicated that miR-483 might regulate genes directly involved in cellular survival and protein expression. Our results highlight that miR-483 is a valuable tool for product-independent engineering of mammalian production cells.

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.

Three-dimensional tumor spheroids for in vitro analysis of bacteria as gene delivery vectors in tumor therapy.

BACKGROUND: Several studies in animal models demonstrated that obligate and facultative anaerobic bacteria of the genera Bifidobacterium, Salmonella, or Clostridium specifically colonize solid tumors. Consequently, these and other bacteria are discussed as live vectors to deliver therapeutic genes to inhibit tumor growth. Therapeutic approaches for cancer treatment using anaerobic bacteria have been investigated in different mouse models. In the present study, solid three-dimensional (3D) multicellular tumor spheroids (MCTS) of the colorectal adenocarcinoma cell line HT-29 were generated and tested for their potential to study prodrug-converting enzyme therapies using bacterial vectors in vitro. RESULTS: HT-29 MCTS resembled solid tumors displaying all relevant features with an outer zone of proliferating cells and hypoxic and apoptotic regions in the core. Upon incubation with HT-29 MCTS, Bifidobacterium bifidum S17 and Salmonella typhimurium YB1 selectively localized, survived and replicated in hypoxic areas inside MCTS. Furthermore, spores of the obligate anaerobe Clostridium sporogenes germinated in these hypoxic areas. To further evaluate the potential of MCTS to investigate therapeutic approaches using bacteria as gene delivery vectors, recombinant bifidobacteria expressing prodrug-converting enzymes were used. Expression of a secreted cytosine deaminase in combination with 5-fluorocytosine had no effect on growth of MCTS due to an intrinsic resistance of HT-29 cells to 5-fluorouracil, i.e. the converted drug. However, a combination of the prodrug CB1954 and a strain expressing a secreted chromate reductase effectively inhibited MCTS growth. CONCLUSIONS: Collectively, the presented results indicate that MCTS are a suitable and reliable model to investigate live bacteria as gene delivery vectors for cancer therapy in vitro.

Unveiling the principle of microRNA-mediated redundancy in cellular pathway regulation.

Understanding the multifaceted nature of microRNA (miRNA) function in mammalian cells is still a challenge. Commonly accepted principles of cooperativity and multiplicity of miRNA function imply that individual mRNAs can be targeted by several miRNAs whereas a single miRNA may concomitantly regulate a subset of different genes. However, there is a paucity of information whether multiple miRNAs regulate critical cellular events and thereby acting redundantly. To gain insight into this notion, we conducted an unbiased high-content miRNA screen by individually introducing 1139 miRNA mimics into Chinese hamster ovary (CHO) cells. We discovered that 66% of all miRNAs significantly impacted on proliferation, protein expression, apoptosis and necrosis. In summary, we provide evidence for a substantial degree of redundancy among miRNAs to maintain cellular homeostasis.

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.

Inefficient transcription is a production bottleneck for artificial therapeutic BiTE® proteins.

Antibodies are potent biopharmaceuticals used to treat severe diseases, including cancers. During the past decade, more complex modalities have been developed including bispecific T-cell engager (BiTE®) molecules, e.g. by Amgen. However, non-natural and complex molecule formats often prove to be difficult-to-express (DTE), which is the case for BiTE® molecules. Due to the growing importance of multispecific modalities such as half-life extended (HLE) BiTE® and HLE dual-targeting bispecific T-cell engager (dBiTE) molecules, this artificial class of therapeutic proteins was investigated for molecular bottlenecks in stable production cell lines, by analyzing all relevant steps of recombinant protein production. As a result, drastically reduced intracellular BiTE® molecule-encoding mRNA levels were identified as a potential production bottleneck. Using in vitro transcription (IVT), the transcription rate of the BiTE® molecule-encoding mRNA was identified as the root cause for reduced amounts of intracellular mRNA. In an attempt to improve the transcription rate of a BiTE® molecule, it could be demonstrated that the artificial and special structure of the BiTE® molecule was not the rate limiting step for reduced IVT rate. However, modulation of the primary DNA sequence led to significant improvement of IVT rate. The analyses presented provide insight into the HLE BiTE® / HLE d(BiTE®) class of DTE proteins and perhaps into other classes of DTE proteins, and therefore may lead to identification of further production bottlenecks and optimization strategies to overcome manufacturability challenges associated with various complex therapeutics.