Genome-scale metabolic models describe cellular metabolism with mechanistic detail. Given their high complexity, such models need to be parameterized correctly to yield accurate predictions and avoid ...overfitting. Effective parameterization has been well-studied for microbial models, but it remains unclear for higher eukaryotes, including mammalian cells. To address this, we enumerated model parameters that describe key features of cultured mammalian cells – including cellular composition, bioprocess performance metrics, mammalian-specific pathways, and biological assumptions behind model formulation approaches. We tested these parameters by building thousands of metabolic models and evaluating their ability to predict the growth rates of a panel of phenotypically diverse Chinese Hamster Ovary cell clones. We found the following considerations to be most critical for accurate parameterization: (1) cells limit metabolic activity to maintain homeostasis, (2) cell morphology and viability change dynamically during a growth curve, and (3) cellular biomass has a particular macromolecular composition. Depending on parameterization, models predicted different metabolic phenotypes, including contrasting mechanisms of nutrient utilization and energy generation, leading to varying accuracies of growth rate predictions. Notably, accurate parameter values broadly agreed with experimental measurements. These insights will guide future investigations of mammalian metabolism.
•A panel of parameters are evaluated for their importance in model accuracy.•Parameters describing the cellular objective, biomass and time-course metabolic shifts profoundly affect model accuracy.•Conventional and alternative objective functions for CHO metabolism are compared and explored.
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•Half-sandwich organoruthenium compounds are extensively investigated as cancer treatments.•Bidentate bioactive ligands with a wide variety of donor atom combinations are ...investigated.•Bidentate bioactive ligands endow in some Ru compounds multimodal anticancer activity.•The bioactivity of the organometallics is often determined by the bidentate ligands.•The complexes have been categorized into 5 structural design concepts.
The small molecule anticancer agent cisplatin and its Pt(II) analogs carboplatin and oxaliplatin are widely used to treat a variety of tumorigenic diseases. Despite their structural simplicity, side effects and disadvantages, they are cornerstones of cancer chemotherapy. Several strategies have been pursued to enhance the activity and reduce the side effects of metal-based drugs, for example, to use bioactive ligands that equip them with novel modes of action, enhance delivery, allow for selective activation, create synergistic effects or improve tumor accumulation. Many of these strategies have been developed for or adapted in the design of half-sandwich organoruthenium compounds. For such compounds decorated with bioactive ligands that coordinate monodentately to the Ru center, we have identified five design concepts (Coord. Chem. Rev. 2021, 439, 213890): (i) the bioactive ligand coordinates directly to the Ru center or (ii) after functionalization with a coordinating group, (iii) the ligand(s) and the Ru center solely define the overall shape of the molecule, (iv) the bioactive ligand is released, and (v) the bioactive ligand acts as a vector to the tumor (cell). Herein, we use these five concepts and explore their application to half-sandwich organoruthenium anticancer compounds in which the bioactive ligand is coordinated to the Ru center through a bidentate chelating motif.
•CHO cells with diverse mAb titres show different sensitivity to proteasome inhibitors.•Using proteasome inhibitors during cell pool construction gives enhanced productivity.•Proteasome inhibitors ...may select for cells with particular folding capacity.
Chinese hamster ovary (CHO) cells are the leading mammalian cell expression platform for biotherapeutic recombinant molecules yet some proteins remain difficult to express (DTE) in this, and other, systems. In recombinant cell lines expressing DTE proteins, cellular processes to restore proteostasis can be triggered when the folding and modification capabilities are exceeded, including the unfolded protein response and ER-associated degradation (ERAD) and proteasomal degradation. We therefore investigated whether the proteasome activity of CHO cells was linked to their ability to produce recombinant proteins. We found cell lines with diverse monoclonal antibody (mAb) productivity show different susceptibilities to inhibitors of proteasome activity. Subsequently, we applied selective pressure using proteasome inhibitors on mAb producing cells to determine the impact on cell growth and recombinant protein production, and to apply proteasome selective pressure above that of a metabolic selection marker during recombinant cell pool construction. The presence of proteasome inhibitors during cell pool construction expressing two different model molecules, including a DTE Fc-fusion protein, resulted in the generation of cell pools with enhanced productivity. The increased productivities, and ability to select for higher producing cells, has potential to improve clonal selection during upstream processes of DTE proteins.
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•Pseudo attP sites are transcriptionally active sites for site-specific integration purposes.•Two pseudo attP sites of CHO-K1 cells are successfully targeted via CRISPR/Cas9 and ...compared in terms of level and stability of expression.•The pseudo attP site of chromosome 6 has higher knock-in efficiency and more homogenous expression than the pseudo attP site of chromosome 3.
Chinese hamster ovary (CHO) cells are regarded as a prominent host for manufacturing therapeutic proteins. Although conventional strategies for generating recombinant proteins in CHO cells depend on the random integration of a gene of interest (GOI), these established techniques occasionally result in genetically heterogeneous cell lines, which causes diminished expression of the recombinant proteins in the long run. Production instability can be reduced by SSI and creates stable cell lines with a consistent expression of the GOI. In this experiment, we demonstrate the targeted incorporation of a reporter cassette in two PhiC31 pseudo attP sites of CHO cells exploiting the homology-directed repair (HDR) generated by the CRISPR/Cas9 platform. Genes encoding GFP and puromycin resistance marker were precisely inserted into these loci via CRISPR/Cas9. Stable cell lines were suitably produced following antibiotic selection. Junction PCR and fluorescence assay determined targeted integration and expression homogeneity of the reporter cassette, respectively. Taken together, our results indicate the possibility of these two PhiC31 pseudo attP sites as the target sites for site-specific integration of a transgene mediated by CRISPR/Cas9. Furthermore, higher knock-in efficiency and expression homogeneity was observed in the pseudo attP site associated with chromosome 6 compared to the pseudo attP site from chromosome 3.
Modulation of expression levels of endogenous or recombinant genes can be of great interest for diverse applications, such as the study of genotype-phenotype relationships for a gene of interest, or ...fine-tuning of transcription to determine physiologically relevant effects of gene expression levels. During the last decades, several synthetic biology tools were established to control gene expression in mammalian cells such as Chinese hamster ovary (CHO) cells, one of the most important cell systems for basic research as well as the production of biopharmaceuticals. Here we describe the use of triplex forming oligos (TFOs), short RNA or ssDNA molecules that can bind to the major grove of their target duplex with great specificity, to control transgene expression in CHO cells. For proof of concept, a panel of TFOs with a size of 10–20 nts were designed with the help of the on-line tool Triplexator targeting the viral cytomegalovirus (CMV) promoter/enhancer region controlling the downstream reporter gene hCD4. The effect of TFOs was tested as ssDNA oligos pre-annealed to the promoter/enhancer region in vitro as well as upon endogenous transcription of the TFO as an RNA molecule binding to their target duplex in vivo. Results showed that not only binding of the TFO, but the exact location of triplex formation within the promoter/enhancer is paramount for transcription inhibition. After relieving a binding conflict by introducing a point mutation within the CMV promoter, longer TFOs (26–30 nts) could be designed and analysed. Selected TFOs achieved a reduction in recombinant hCD4 expression of up to 85% in CHO-K1 cells.
•Triplex forming oligo sequences (TFOs) with a size of 10–20 nts were designed to target the viral cytomegalovirus (CMV) promoter/enhancer .•Binding of the TFO, but also the exact location of triplex formation within the promoter/enhancer is paramount for transcription inhibition.•Selected TFOs achieved a reduction in recombinant gene expression of up to 85% in CHO-K1 cells.
Large scale biopharmaceutical production of biologics relies on the overexpression of foreign proteins by cells cultivated in stirred tank bioreactors. It is well recognized and documented fact that ...protein overexpression may impact host cell metabolism and that factors associated with large scale culture, such as the hydrodynamic forces and inhomogeneities within the bioreactors, may promote cellular stress. The metabolic adaptations required to support the high‐level expression of recombinant proteins include increased energy production and improved secretory capacity, which, in turn, can lead to a rise of reactive oxygen species (ROS) generated through the respiration metabolism and the interaction with media components. Oxidative stress is defined as the imbalance between the production of free radicals and the antioxidant response within the cells. Accumulation of intracellular ROS can interfere with the cellular activities and exert cytotoxic effects via the alternation of cellular components. In this context, strategies aiming to alleviate oxidative stress generated during the culture have been developed to improve cell growth, productivity, and reduce product microheterogeneity. In this review, we present a summary of the different approaches used to decrease the oxidative stress in Chinese hamster ovary cells and highlight media development and cell engineering as the main pathways through which ROS levels may be kept under control.
Reactive oxygen species are generated during recombinant protein production using CHO cells and can lead to oxidative stress. Consequently, cell growth, productivity and product quality can be negatively impacted. In order to alleviate oxidative stress, two main strategies can be used: media development and cell engineering. This review gives an overview of the different approaches available in literature.
Much of the biopharmaceutical industry's success over the past 30 years has relied on products derived from Chinese Hamster Ovary (CHO) cell lines. During this time, improvements in mammalian cell ...cultures have come from cell line development and process optimization suited for large-scale fed-batch processes. Originally developed for high cell densities and sensitive products, perfusion processes have a long history. Driven by high volumetric titers and a small footprint, perfusion-based bioprocess research has regained an interest from academia and industry. The recent pandemic has further highlighted the need for such intensified biomanufacturing options. In this review, we outline the technical history of research in this field as it applies to biologics production in CHO cells. We demonstrate a number of emerging trends in the literature and corroborate these with underlying drivers in the commercial space. From these trends, we speculate that the future of perfusion bioprocesses is bright and that the fields of media optimization, continuous processing, and cell line engineering hold the greatest potential. Aligning in its continuous setup with the demands for Industry 4.0, perfusion biomanufacturing is likely to be a hot topic in the years to come.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Chinese hamster ovary (CHO) cells are the preferred mammalian host for the large-scale production of recombinant proteins in the biopharmaceutical industry. Research endeavors have been directed to ...the optimization of CHO-based bioprocesses to increase protein quantity and quality, often in an empirical manner. To provide a rationale for those achievements, a myriad of CHO proteomic studies has arisen in recent decades. This review gives an overview of significant advances in LC-MS-based proteomics and sheds light on CHO proteomic studies, with a particular focus on CHO cells with superior bioprocessing phenotypes (growth, viability, titer, productivity and cQA), that have exploited novel proteomic or sub-omic techniques. These proteomic findings expand the current knowledge and understanding about the underlying protein clusters, protein regulatory networks and biological pathways governing such phenotypic changes. The proteomic studies, highlighted herein, will help in the targeted modulation of these cell factories to the desired needs.
•Proteomic and sub-omic studies on CHO cells reveal a holistic picture of bottlenecks in recombinant protein production.•Data- and knowledge-driven design of cell factories with improved bioprocessing phenotypes.•Advances in the proteomics pipeline push the boundaries towards greater proteomic depths.
A new area of focus in Chinese hamster ovary (CHO) biotechnology is the role of small (exosomes) and large (microvesicles or microparticles) extracellular vesicles (EVs). CHO cells in culture ...exchange large quantities of proteins and RNA through these EVs, yet the content and role of these EVs remain elusive. MicroRNAs (miRs or miRNA) are central to adaptive responses to stress and more broadly to changes in culture conditions. Given that EVs are highly enriched in miRs, and that EVs release large quantities of miRs both in vivo and in vitro, EVs and their miR content likely play an important role in adaptive responses. Here we report the miRNA landscape of CHO cells and their EVs under normal culture conditions and under ammonia and osmotic stress. We show that both cells and EVs are highly enriched in five miRs (among over 600 miRs) that make up about half of their total miR content, and that these highly enriched miRs differ significantly between normal and stress culture conditions. Notable is the high enrichment in miR‐92a and miR‐23a under normal culture conditions, in contrast to the high enrichment in let‐7 family miRs (let‐7c, let‐7b, and let‐7a) under both stress conditions. The latter suggests a preserved stress‐responsive function of the let‐7 miR family, one of the most highly preserved miR families across species, where among other functions, let‐7 miRs regulate core oncogenes, which, depending on the biological context, may tip the balance between cell cycle arrest and apoptosis. While the expected—based on their profound enrichment—important role of these highly enriched miRs remains to be dissected, our data and analysis constitute an important resource for exploring the role of miRs in cell adaptation as well as for synthetic applications.
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