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.

Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates.

pH-sensitive fluorescent proteins as genetically encoded pH sensors are promising tools for monitoring intra- and extracellular pH. However, there is a lack of ratiometric pH sensors, which offer a good dynamic range and can be purified and applied extracellularly to investigate uptake. In our study, the bright fluorescent protein CoGFP_V0 was C-terminally fused to the ligand epidermal growth factor (EGF) and retained its dual-excitation and dual-emission properties as a purified protein. The tandem fluorescent variants EGF-CoGFP-mTagBFP2 (pK' = 6.6) and EGF-CoGFP-mCRISPRed (pK' = 6.1) revealed high dynamic ranges between pH 4.0 and 7.5. Using live-cell fluorescence microscopy, both pH sensor molecules permitted the conversion of fluorescence intensity ratios to detailed intracellular pH maps, which revealed pH gradients within endocytic vesicles. Additionally, extracellular binding of the pH sensors to cells expressing the EGF receptor (EGFR) enabled the tracking of pH shifts inside cultivation chambers of a microfluidic device. Furthermore, the dual-emission properties of EGF-CoGFP-mCRISPRed upon 488 nm excitation make this pH sensor a valuable tool for ratiometric flow cytometry. This high-throughput method allowed for the determination of internalization rates, which represents a promising kinetic parameter for the in vitro characterization of protein-drug conjugates in cancer therapy.

Hyperosmolality in CHO cell culture: effects on the proteome.

Chinese hamster ovary (CHO) cells are the most commonly used host cell lines for therapeutic protein production. Exposure of these cells to highly concentrated feed solution during fed-batch cultivation can lead to a non-physiological increase in osmolality (> 300 mOsm/kg) that affects cell physiology, morphology, and proteome. As addressed in previous studies (and indeed, as recently addressed in our research), hyperosmolalities of up to 545 mOsm/kg force cells to abort proliferation and gradually increase their volume-almost tripling it. At the same time, CHO cells also show a significant hyperosmolality-dependent increase in mitochondrial activity. To gain deeper insight into the molecular mechanisms that are involved in these processes, as detailed in this paper, we performed a comparative quantitative label-free proteome study of hyperosmolality-exposed CHO cells compared with control cells. Our analysis revealed differentially expressed key proteins that mediate mitochondrial activation, oxidative stress amelioration, and cell cycle progression. Our studies also demonstrate a previously unknown effect: the strong regulation of proteins can alter both cell membrane stiffness and permeability. For example, we observed that three types of septins (filamentous proteins that form diffusion barriers in the cell) became strongly up-regulated in response to hyperosmolality in the experimental setup. Overall, these new observations correlate well with recent CHO-based fluxome and transcriptome studies, and reveal additional unknown proteins involved in the response to hyperosmotic pressure by over-concentrated feed in mammalian cells.Key points• First-time comparative proteome analysis of CHO cells exposed to over-concentrated feed.• Discovery of membrane barrier-forming proteins up-regulation under hyperosmolality.• Description of mitochondrial and protein chaperones activation in treated cells.

Human Coxsackie- and adenovirus receptor is a putative target of neutrophil elastase-mediated shedding.

BACKGROUND: During viral-induced myocarditis, immune cells migrate towards the site of infection and secrete proteases, which in turn can act as sheddases by cleaving extracellular domains of transmembrane proteins. We were interested in the shedding of the Coxsackie- and adenovirus receptor (CAR) that acts as an entry receptor for both eponymous viruses, which cause myocarditis. CAR shedding by secreted immune proteases could result in a favourable outcome of myocarditis as CAR's extracellular domain would be removed from the cardiomyocytes' surface leading to decreased susceptibility to ongoing viral infections. METHODS AND RESULTS: In this work, matrix metalloproteinases and serine proteinases were screened for their proteolytic activity towards human CAR. Whereas matrix metalloproteinases, proteinase 3, and cathepsin G did not cleave human recombinant CAR or only within long incubation times, neutrophil elastase showed a distinct cleavage pattern of CAR's extracellular domain that was time- and dose-dependent. Neutrophil elastase cleaves CAR at its membrane-proximal immunoglobulin domain as we determined by nanoLC-MS/MS. Furthermore, neutrophil elastase treatment of cells reduced CAR surface levels as seen by flow cytometry and immunofluorescence microscopy. CONCLUSIONS: With this study, we show that CAR might be a target for shedding by neutrophil elastase.

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.

A positive pressure workstation for semi-automated peptide purification of complex proteomic samples.

RATIONALE: High-throughput reliable data generation has become a substantial requirement in many "omics" investigations. In proteomics the sample preparation workflow consists of multiple steps adding more bias to the sample with each additional manual step. Especially for label-free quantification experiments, this drastically impedes reproducible quantification of proteins in replicates. Here, a positive pressure workstation was evaluated to increase automation of sample preparation and reduce workload as well as consumables. METHODS: Digested peptide samples were purified utilizing a new semi-automated sample preparation device, the Resolvex A200, followed by nanospray liquid chromatography/electrospray ionization (nLC/ESI) Orbitrap tandem mass spectrometry (MS/MS) measurements. In addition, the sorbents Maestro and WWP2 (available in conventional cartridge and dual-chamber narrow-bore extraction columns) were compared with Sep-Pak C18 cartridges. Raw data was analyzed by MaxQuant and Perseus software. RESULTS: The semi-automated workflow with the Resolvex A200 workstation and both new sorbents produced highly reproducible results within 10-300 µg of peptide starting material. The new workflow performed equally as well as the routinely conducted manual workflow with similar technical variability in MS/MS-based identifications of peptides and proteins. A first application of the system to a biological question contributed to highly reliable results, where time-resolved proteomic data was separated by principal component analysis (PCA) and hierarchical clustering. CONCLUSIONS: The new workstation was successfully established for proteolytic peptide purification in our proteomic workflow without any drawbacks. Highly reproducible results were obtained in decreased time per sample, which will facilitate further large-scale proteomic investigations.

Clonal variations in CHO IGF signaling investigated by SILAC-based phosphoproteomics and LFQ-MS.

Chinese hamster ovary (CHO) cells are commonly used for the production of monoclonal antibodies. Omics technologies have been used to elucidate cellular switch points which result in higher monoclonal antibody (mAb) productivity and process yields in CHO and other biopharmaceutical production cell lines such as human or mouse. Currently, investigations of the phosphoproteome in CHO cell lines are rare yet could provide further insights into cellular mechanisms related to target product expression. Therefore, we investigated CHO IGF-signaling events using a comparative expression and phosphoproteomic approach in recombinant mAb-producing XL99 cell lines and corresponding parental strain. Differences were found on the level of protein expression between producer and parental cells in the exponential growth phase, mainly in proteins related to the lysosome, oligosaccharide metabolic processes, stress response, and cellular homeostasis. Within a stable isotope labeling by amino acids in cell culture (SILAC)-based phosphoproteomic investigation of IGF signaling, expected general regulation of phosphorylation sites and cell line-specific responses were observed. Detected early phosphorylation events can be associated to observed effects of IGF on cellular growth, metabolism, and cell cycle distribution. Producer cell line-specific signaling exhibited differences to parental cells in intracellular trafficking and transcriptional processes, along with an overall lower amount of observable cross talk to other signaling pathways. By combining label-free and SILAC-based expression for phosphoproteomic analyses, cellular differences in the highly interactive levels of signaling and protein expression were detected, indicating alterations in metabolism and growth following treatment with an exogenous growth factor. The characterization of cell lines and effects of IGF addition resulted in identification of metabolic switch points. With this data, it will be possible to modulate pathways towards increased CHO process yield by targeted application of small-molecule inhibitors.

Integrative analysis of DNA methylation and gene expression in butyrate-treated CHO cells.

The cellular mechanisms responsible for the versatile properties of CHO cells as the major production cell line for biopharmaceutical molecules are not entirely understood yet, although several 'omics' data facilitate the understanding of CHO cells and their reactions to environmental conditions. However, genome-wide studies of epigenetic processes such as DNA methylation are still limited. To prove the applicability and usefulness of integrating DNA methylation and gene expression data in a biotechnological context, we exemplarily analyzed the time course of cellular reactions upon butyrate addition in antibody-producing CHO cells by whole-genome bisulfite sequencing and CHO-specific cDNA microarrays. Gene expression and DNA methylation analyses showed that pathways known to be affected by butyrate, including cell cycle and apoptosis, as well as pathways potentially involved in butyrate-induced hyperproductivity such as central energy metabolism and protein biosynthesis were affected. Differentially methylated regions were furthermore found to contain binding-site motifs of specific transcription factors and were hypothesized to represent regulatory regions closely connected to the cellular response to butyrate. Generally, our experiment underlines the benefit of integrating DNA methylation and gene expression data, as it provided potential novel candidate genes for rational cell line development and allowed for new insights into the butyrate effect on CHO cells.

Process boundaries of irreversible scCO2 -assisted phase separation in biphasic whole-cell biocatalysis.

The formation of stable emulsions in biphasic biotransformations catalyzed by microbial cells turned out to be a major hurdle for industrial implementation. Recently, a cost-effective and efficient downstream processing approach, using supercritical carbon dioxide (scCO2 ) for both irreversible emulsion destabilization (enabling complete phase separation within minutes of emulsion treatment) and product purification via extraction has been proposed by Brandenbusch et al. (2010). One of the key factors for a further development and scale-up of the approach is the understanding of the mechanism underlying scCO2 -assisted phase separation. A systematic approach was applied within this work to investigate the various factors influencing phase separation during scCO2 treatment (that is pressure, exposure of the cells to CO2 , and changes of cell surface properties). It was shown that cell toxification and cell disrupture are not responsible for emulsion destabilization. Proteins from the aqueous phase partially adsorb to cells present at the aqueous-organic interface, causing hydrophobic cell surface characteristics, and thus contribute to emulsion stabilization. By investigating the change in cell-surface hydrophobicity of these cells during CO2 treatment, it was found that a combination of catastrophic phase inversion and desorption of proteins from the cell surface is responsible for irreversible scCO2 mediated phase separation. These findings are essential for the definition of process windows for scCO2 -assisted phase separation in biphasic whole-cell biocatalysis.

The DNA methylation landscape of Chinese hamster ovary (CHO) DP-12 cells.

Chinese hamster ovary (CHO) cells represent the most commonly used production cell line for therapeutic proteins. By recent genome and transcriptome sequencing a basis was created for future investigations of genotype-phenotype relationships and for improvement of CHO cell productivity and product quality. In this context information is missing about DNA cytosine methylation as a crucial epigenetic modification and an important element in mammalian genome regulation and development. Here, we present the first DNA methylation map of a CHO cell line in single-base resolution that was generated by whole genome bisulfite sequencing combined with gene expression analysis by CHO microarrays. We show CHO DP-12 cells to exhibit global hypomethylation compared to a majority of mammalian methylomes and hypermethylation of CpG-dense regions at gene promoters called CpG islands. We also observed partially methylated domains that cover 62% of the CHO DP-12 cell genome and contain functional clusters of genes. Gene expression analysis showed these clusters to be either highly or weakly expressed with regard to CHO-specific characteristics and hence proves DNA methylation in CHO cells to be an important link between genomics and transcriptomics.

2D-DIGE screening of high-productive CHO cells under glucose limitation--basic changes in the proteome equipment and hints for epigenetic effects.

CHO derivates (Chinese hamster ovary) belong to the most important mammalian cells for industrial recombinant protein production. Many efforts have been made to improve productivity and stability of CHO cells in bioreactor processes. Here, we followed up one barely understood phenomenon observed with process optimizations: a significantly increased cell-specific productivity in late phases of glucose-limited perfusion cultivations, when glucose (and lactate) reserves are exhausted. Our aim was to elucidate the cellular activities connected to the metabolic shift from glucose surplus to glucose limitation phase. With 2D-DIGE, we compared three stages in a perfusion culture of CHO cells: the initial growth with high glucose concentration and low lactate production, the second phase with glucose going to limitation and high lactate level, and finally the state of glucose limitation and also low lactate concentration but increased cell-specific productivity. With our proteomic approach we were able to demonstrate consequences of glucose limitation for the protein expression machinery which also could play a role for a higher recombinant protein production. Most interestingly, we detected epigenetic effects on the level of proteins involved in histone modification (HDAC1/-2, SET, RBBP7, DDX5). Together with shifts in the protein inventory of energy metabolism, cytoskeleton and protein expression, a picture emerges of basic changes in the cellular equipment under long-term glucose limitation of CHO cells.

Establishment of a CpG island microarray for analyses of genome-wide DNA methylation in Chinese hamster ovary cells.

Optimizing productivity and growth rates of recombinant Chinese hamster ovary (CHO) cells requires insight into the regulation of cellular processes. In this regard, the elucidation of the epigenetic process of DNA methylation, known to influence transcription by a differential occurrence in CpG islands in promoter regions, is increasingly gaining importance. However, DNA methylation has not yet been investigated on a genomic scale in CHO cells and suitable tools have not existed until now. Based on the genomic and transcriptomic CHO data currently available, we developed a customized oligonucleotide microarray covering 19598 CpG islands (89 % of total bioinformatically identified CpG islands) in the CHO genome. We applied our CHO-specific CpG island microarray to investigate the effect of butyrate treatment on differential DNA methylation in CHO cultures in a time-dependent approach. Supplementation of butyrate is known to enhance cell specific productivities in CHO cells and leads to alterations of epigenetic silencing events. Gene ontology clusters regarding, e.g., chromatin modification or DNA repair, were significantly overrepresented 24 h after butyrate addition. Functional classifications furthermore indicated that several major signaling systems such as the Wnt/ß-catenin pathway were affected by butyrate treatment. Our novel CHO-specific CpG island microarray will provide valuable information in future studies of cellular processes associated with productivity and product characteristics.

Utilization and evaluation of CHO-specific sequence databases for mass spectrometry based proteomics.

Recently released sequence information on Chinese hamster ovary (CHO) cells promises to not only facilitate our understanding of these industrially important cell factories through direct analysis of the sequence, but also to enhance existing methodologies and allow new tools to be developed. In this article we demonstrate the utilization of CHO specific sequence information to improve mass spectrometry (MS) based proteomic identification. The use of various CHO specific databases enabled the identification of 282 additional proteins, thus increasing the total number of identified proteins by 40-50%, depending on the sample source and methods used. In addition, a considerable portion of those proteins that were identified previously based on inter-species sequence homology were now identified by a larger number of peptides matched, thus increasing the confidence of identification. The new sequence information offers improved interpretation of proteomic analyses and will, in the years to come, prove vital to unraveling the CHO proteome.

Interferon-beta modulates protein synthesis in the central nervous system.

Interferon-beta (IFN-beta), acting canonically via the modulation of transcription, affects neocortical pyramidal neurons. By use of 2-D differential gel electrophoresis and subsequent mass spectrometry we identified IFN-beta regulated proteins in the central nervous system. These proteins are involved in cytoskeleton assembly, protein transport and nucleotide metabolism and, as such, serve regenerative and protective functions. Electrophysiologically, IFN-beta mediated protein synthesis is essential for part of the excitatory neuronal effects, as revealed under blockade of protein biosynthesis. This study presents novel effects of IFN-beta in the central nervous system and begins to unravel the mechanism behind the known excitability changes in neurons.

Neuroproteomics in stem cell differentiation.

The term "proteome" is used to describe the entire complement of proteins in a given organism or in a system at a given time. Proteome analysis in neuroscience, also called "neuroproteomics" or "neuromics" is in its initial stage, and shows a deficit of studies in the context of brain development. It is the main objective of this review to illustrate the potential of neuroproteomics as a tool to unravel the differentiation of neural stem or progenitor cells to terminally differentiated neurons. Experimental results regarding the rat striatal progenitor model cell line ST14A are presented to illustrate the large rearrangements of the proteome during the differentiation process of neural progenitor cells and their modification by neurotrophic factors like the glial cell line-derived neurotrophic factor (GDNF). Thereby native stem cells and cells transfected with GDNF gene were investigated at the proliferative state and at seven time points up to 72 h after induction of differentiation. In addition, the immortalized human fetal midbrain stem cell line ReNcell VM was analyzed in order to detect stem cell differentiation associated changes of the protein profile. This review gives also an outlook on technical improvements and perspectives of application of neural stem cell proteomics.

2-DE profiling of GDNF overexpression-related proteome changes in differentiating ST14A rat progenitor cells.

Targeted differentiation of neural progenitor cells (NPCs) is a challenge for treatment of neurodegenerative diseases by cell replacement therapy and cell signalling manipulation. Here, we applied a proteome profiling approach to the rat striatal progenitor model cell line ST14A in order to elucidate cellular differentiation processes. Native cells and cells transfected with the glial cell line-derived neurotrophic factor (GDNF) gene were investigated at the proliferative state and at seven time points up to 72 h after induction of differentiation. 2-DE combined with MALDI-MS was used to create a reference 2-DE-map of 652 spots of which 164 were identified and assigned to 155 unique proteins. For identification of protein expression changes during cell differentiation, spot patterns of triplicate gels were matched to the 2-DE-map. Besides proteins that display expression changes in native cells, we also noted 43 protein-spots that were differentially regulated by GDNF overexpression in more than four time points of the experiment. The expression patterns of putative differentiation markers such as annexin 5 (ANXA5), glucosidase II beta subunit (GLU2B), phosphatidylethanolamine-binding protein 1 (PEBP1), myosin regulatory light chain 2-A (MLRA), NASCENT polypeptide-associated complex alpha (NACA), elongation factor 2 (EF2), peroxiredoxin-1 (PRDX1) and proliferating cell nuclear antigen (PCNA) were verified by Western blotting. The results reflect the large rearrangements of the proteome during the differentiation process of NPCs and their strong modification by neurotrophic factors like GDNF.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitor Atorvastatin mediated effects depend on the activation status of target cells in PLP-EAE.

The effect of Atorvastatin on transcriptional activity in murine experimental autoimmune encephalomyelitis (EAE) induced by PLP peptide 139-151 was analyzed by DNA microarray technique in lymph nodes and spinal cord at onset (10 days), height (20 days) and first remission (30 days) of disease. Fourteen genes were selectively influenced by Atorvastatin in EAE mice. They are mainly related to immune cell functions and regulation of cell-to-cell interaction. Interestingly, seven genes were also differentially regulated in CFA-injected control mice. But qualitative and quantitative differences to EAE mice argue for a dependency of statin effects on the activation status of target cells. Differential regulation of the newly detected candidate genes of statin effects COX-1 and HSP-105 and the previously known statin-responsive genes ICAM-1 and CD86 was confirmed by Western blot and immunohistochemistry. Flow cytometric analysis of lymph node cells revealed that the effect of Atorvastatin treatment in non-immunized healthy animals resembled the effect of immunization with PLP peptide regarding changes of T helper cells, activated B cells and macrophages. In EAE mice, these effects were partially reversed by Atorvastatin treatment. Monitoring of expression of the newly identified candidate genes and patterns of lymphocyte subpopulations might predict the responsiveness of multiple sclerosis patients to statin treatment.

2-DE proteomic profiling of neuronal stem cells.

Proteomics has become a powerful tool in neuroscience studies. Although numerous human neural stem cells are available for research purposes since many years, there exists only limited information on proteomic data from stable neural stem cell lines. Profiling and functional proteome studies of neuronal stem cells will help to describe the protein inventory as well as protein activity and interactions, subcellular localization and posttranslational modifications. The proteomic analysis of neuronal differentiation processes will elucidate the complex events leading to the generation of different phenotypes via distinctive developmental programs that control self-renewal, differentiation, and plasticity. Using the ReNcell VM197 model, a cell line derived from human fetal ventral mesencephalon stem cells, we studied the protein inventory of the stem cells by 2-DE gel electrophoresis and mass spectrometric protein identification and constructed a 2-DE protein map consisting of more than 400 identified protein spots. This proteome reference database constitutes the basis for further investigations of differential protein expression during differentiation. A profiling of the neuronal differentiation-associated changes displayed the large rearrangement of the proteome during this process, and the proteomic techniques proved to be a valuable tool for the elucidation of neuronal differentiation process and for target protein screening.

2-DE proteome analysis of a proliferating and differentiating human neuronal stem cell line (ReNcell VM).

The proteome of a proliferating human stem cell line was analyzed and then utilized to detect stem cell differentiation-associated changes in the protein profile. The analysis was conducted with a stable human fetal midbrain stem cell line (ReNcell VM) that displays the properties of a neural stem cell. Therefore, acquisition of proteomic data should be representative of cultured human neural stem cells (hNSCs) in general. Here we present a 2-DE protein-map of this cell line with annotations of 402 spots representing 318 unique proteins identified by MS. The subsequent proteome profiling of differentiating cells of this stem cell line at days 0, 4 and 7 of differentiation revealed changes in the expression of 49 identified spots that could be annotated to 45 distinct proteins. This differentiation-associated expression pattern was validated by Western blot analysis for transgelin-2, proliferating cell nuclear antigen, as well as peroxiredoxin 1 and 4. The group of regulated proteins also included NudC, ubiquilin-1, STRAP, stress-70 protein, creatine kinase B, glial fibrillary acidic protein and vimentin. Our results reflect the large rearrangement of the proteome during the differentiation process of the stem cells to terminally differentiated neurons and offer the possibility for further characterization of specific targets driving the stem cell differentiation.