Publications

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chromatographic purification of recombinant human erythropoietin.recombinant human erythropoietin is a valuable therapeutic protein used in the treatment of several serious diseases. it exists in different isoforms and is produced by genetically modified mammalian cells such as chinese hamster ovary or human embryonic kidney cells. as for other biopharmaceutical drugs, a key factor for its successful industrial production is to achieve a high degree of purity and to decrease the content of critical impurities to trace amounts. this goal is achieved in the sep ...201930810853
novel column generation-based optimization approach for poly-pathway kinetic model applied to cho cell culture.mathematical modelling can provide precious tools for bioprocess simulation, prediction, control and optimization of mammalian cell-based cultures. in this paper we present a novel method to generate kinetic models of such cultures, rendering complex metabolic networks in a poly-pathway kinetic model. the model is based on subsets of elementary flux modes (efms) to generate macro-reactions. thanks to our column generation-based optimization algorithm, the experimental data are used to identify t ...201930809468
systematic evaluation of site-specific recombinant gene expression for programmable mammalian cell engineering.many branches of biology depend on stable and predictable recombinant gene expression, which has been achieved in recent years through targeted integration of the recombinant gene into defined integration sites. however, transcriptional levels of recombinant genes in characterized integration sites are controlled by multiple components of the integrated expression cassette. lack of readily available tools has inhibited meaningful experimental investigation of the interplay between the integratio ...201930807689
model-assisted design of experiments as a concept for knowledge-based bioprocess development.design of experiments methods offer systematic tools for bioprocess development in quality by design, but their major drawback is the user-defined choice of factor boundary values. this can lead to several iterative rounds of time-consuming and costly experiments. in this study, a model-assisted design of experiments concept is introduced for the knowledge-based reduction of boundary values. first, the parameters of a mathematical process model are estimated. second, the investigated factor comb ...201930806781
crispr/cas9-mediated knockout of microrna-744 improves antibody titer of cho production cell lines.micrornas (mirnas) are noncoding rnas that serve as versatile molecular engineering tools to improve production cells by overexpression or knockdown of mirnas showing beneficial or adverse effects on cell-culture performance. the genomic knockout (ko) of noncoding rnas in chinese hamster ovary (cho) production cells has not been reported. however, given the significant number of mirnas showing negative effects on cho-bioprocess performance and the development of clustered regularly interspaced s ...201930802343
transcriptomic analysis of igg4 fc-fusion protein degradation in a panel of clonally-derived cho cell lines using rnaseq.in this study, we report an investigation of a panel of clonally-derived chinese hamster ovary (cho) cell lines exhibiting variability in the proportion of full-length igg4 fc-fusion protein produced. the recombinant protein was found to be degraded during cell culture into four shorter "clipped" species (three of the four cleavage sites occurred at arginine residues) and preliminary analyses suggested that a host cell enzyme was responsible for proteolysis. to identify the specific enzyme respo ...201930802296
model-based optimization of antibody galactosylation in cho cell culture.exerting control over the glycan moieties of antibody therapeutics is highly desirable from a product safety and batch-to-batch consistency perspective. strategies to improve antibody productivity may compromise quality, while interventions for improving glycoform distribution can adversely affect cell growth and productivity. process design therefore needs to consider the trade-off between preserving cellular health and productivity while enhancing antibody quality. in this work, we present a m ...201930802295
pmab-213: a monoclonal antibody for immunohistochemical analysis against pig podoplanin.podoplanin (pdpn) is known to be expressed in normal tissues, including lymphatic endothelial cells, renal podocytes, and type i lung alveolar cells. monoclonal antibodies (mabs) against human, mouse, rat, rabbit, dog, cat, and bovine pdpn have already been established; however, mabs against pig pdpn (ppdpn) are lacking. in the present study, mice were immunized with ppdpn-overexpressing chinese hamster ovary (cho)-k1 cells (cho/ppdpn), and hybridomas producing mabs against ppdpn were identified ...201930802179
enhanced cho clone screening: application of targeted locus amplification and next-generation sequencing technologies for cell line development.early analytical clone screening is important during chinese hamster ovary (cho) cell line development of biotherapeutic proteins to select a clonally derived cell line with most favorable stability and product quality. sensitive sequence confirmation methods using mass spectrometry have limitations in throughput and turnaround time. next-generation sequencing (ngs) technologies emerged as alternatives for cho clone analytics. we report an efficient ngs workflow applying the targeted locus ampli ...201930793505
pmab-219: a monoclonal antibody for the immunohistochemical analysis of horse podoplanin.monoclonal antibodies (mabs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (pdpn), a lymphatic endothelial cell marker, have been established in our previous studies. however, mabs against horse pdpn (horpdpn), which are useful for immunohistochemical analysis, remain to be developed. in the present study, mice were immunized with horpdpn-overexpressing chinese hamster ovary (cho)-k1 cells (cho/horpdpn), and hybridomas producing mabs against horpdpn were screened using flow ...201930766925
challenging the workhorse: comparative analysis of eukaryotic micro-organisms for expressing monoclonal antibodies.for commercial protein therapeutics, chinese hamster ovary (cho) cells have an established history of safety, proven capability to express a wide range of therapeutic proteins and high volumetric productivities. expanding global markets for therapeutic proteins and increasing concerns for broadened access of these medicines has catalyzed consideration of alternative approaches to this platform. reaching these objectives likely will require an order of magnitude increase in volumetric productivit ...201930739333
an integrated platform for mucin-type o-glycosylation network generation and visualization.mucin-type o-glycans have profound effects on the structure and stability of glycoproteins. o-glycans on the cell surface proteins also modulate the cell's interactions with the surrounding environments and other cells. the synthetic pathway of o-glycans involves a large number of enzymes with diverse substrate specificity. the expression pattern of these enzymes is cell and tissue-specific, thus making the pathway highly diverse. to facilitate pathway analysis in a cell and tissue-specific fash ...201930739313
a comparison of the ability of leu8- and pro8-oxytocin to regulate intracellular ca2+ and ca2+-activated k+ channels at human and marmoset oxytocin receptors.the neurohypophyseal hormone oxytocin (ot) regulates biologic functions in both peripheral tissues and the central nervous system. in the central nervous system, ot influences social processes, including peer relationships, maternal-infant bonding, and affiliative social relationships. in mammals, the nonapeptide ot structure is highly conserved with leucine in the eighth position (leu8-ot). in marmosets (callithrix), a nonsynonymous nucleotide substitution in the oxt gene codes for proline in t ...201930739093
study of the mechanism for increased protein expression via transcription potency reduction of the selection marker.stable transfection of mammalian cells using various expression cassettes for exogenous gene expression has been well established. the impact of critical factors in these cassettes, such as promoter and enhancer elements, on recombinant protein production in mammalian cells has been studied extensively to optimize the expression efficiency. however, few studies on the correlation between the strength of selection marker and the expression of gene of interest (goi) have been reported. here we inv ...201930730009
free, unlinked glycosylphosphatidylinositols on mammalian cell surfaces revisited.glycosylphosphatidylinositols (gpis) are linked to many cell-surface proteins, anchor these proteins in the membrane, and are well characterized. however, gpis that exist in the free form on the mammalian cell surface remain largely unexplored. to investigate free gpis in cultured cell lines and mouse tissues, here we used the t5-4e10 mab (t5 mab), which recognizes unlinked gpis having an n-acetylgalactosamine (galnac) side chain linked to the first mannose at the nonreducing terminus. we detect ...201930728244
selective inhibition of par4 (protease-activated receptor 4)-mediated platelet activation by a synthetic nonanticoagulant heparin analog.objective- par4 (protease-activated receptor 4), one of the thrombin receptors in human platelets, has emerged as a promising target for the treatment of arterial thrombotic disease. previous studies implied that thrombin exosite ii, known as a binding site for heparin, may be involved in thrombin-induced par4 activation. in the present study, a heparin octasaccharide analog containing the thrombin exosite ii-binding domain of heparin was chemically synthesized and investigated for anti-par4 eff ...201930727756
c3p3-g1: first generation of a eukaryotic artificial cytoplasmic expression system.most eukaryotic expression systems make use of host-cell nuclear transcriptional and post-transcriptional machineries. here, we present the first generation of the chimeric cytoplasmic capping-prone phage polymerase (c3p3-g1) expression system developed by biological engineering, which generates capped and polyadenylated transcripts in host-cell cytoplasm by means of two components. first, an artificial single-unit chimeric enzyme made by fusing an mrna capping enzyme and a dna-dependent rna pol ...201930726994
enhancing the functionality of a microscale bioreactor system as an industrial process development tool for mammalian perfusion culture.without a scale-down model for perfusion, high resource demand makes cell line screening or process development challenging, therefore, potentially successful cell lines or perfusion processes are unrealized and their ability untapped. we present here the refunctioning of a high-capacity microscale system that is typically used in fed-batch process development to allow perfusion operation utilizing in situ gravity settling and automated sampling. in this low resource setting, which involved rout ...201930712286
mycobacterium tuberculosis ag85b:hfcγ1 recombinant fusion protein as a selective receptor-dependent delivery system for antigen presentation.introducing an effective vaccine for tuberculosis (tb) is an urgent need. mycobacterium tuberculosis (mtb) ag85 complex is suggested for making protective immunodominant antigens for design and development of novel tb vaccine. in the present study, a pdr2ef1-fcγ1 vector has been used to make ag85b:hfcγ1 recombinant fusion protein. briefly, specific xbai and noti site incorporated primers were used to amplify mtb-fbpb gene by pcr, ta-cloned and amplified in e.coli dh5α. the resulting vector then ...201930711546
case study: the characterization and implementation of dielectric spectroscopy (biocapacitance) for process control in a commercial gmp cho manufacturing process.dielectric spectroscopy (biocapacitance) is an up-and-coming technology for real time monitoring of biomass in cell culture processes and has opened the door for next-generation cell culture process control techniques such as automated on-demand nutrient feeding. in this case study we empirically demonstrate the lower limit of quantitation (loq), probe-to-probe consistency, and scalability of in situ biocapacitance probes using data generated from small- and large-scale chinese hamster ovary (ch ...201930707503
dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate-independent manner.the human dopamine transporter (hdat) regulates the level of the neurotransmitter dopamine (da) in the synaptic cleft and recycles da for storage in the presynaptic vesicular pool. many neurotransmitter transporters exist as oligomers, but the physiological role of oligomerization remains unclear; for example, it has been speculated to be a prerequisite for amphetamine-induced release and protein trafficking. previous studies point to an oligomeric quaternary structure of hdat; however, the exac ...201930705091
slc20a2 variants cause dysfunctional phosphate transport activity in endothelial cells induced from idiopathic basal ganglia calcification patients-derived ipscs.idiopathic basal ganglia calcification (ibgc) is a rare neuropsychiatric illness also known as fahr's disease or primary familial brain calcification (pfbc). ibgc is caused by slc20a2 variants, which encodes the inorganic phosphate (pi) transporter pit-2, a transmembrane protein associated with pi homeostasis. we have reported novel slc20a2 variants in the japanese population and established an induced pluripotent stem cells (ipscs) from an ibgc patient carrying a slc20a2 variant. to investigate ...201930704756
cho genome mining for synthetic promoter design.synthetic promoters are an attractive alternative for use in mammalian hosts such as cho cells as they can be designed de novo with user-defined functionalities. in this study, we describe and validate a method for bioprocess-directed design of synthetic promoters utilizing cho genomic sequence information. we designed promoters with two objective features, (i) constitutive high-level recombinant gene transcription, and (ii) upregulated transcription under mild hypothermia or late-stage culture. ...201930703471
comparison of data-acquisition methods for the identification and quantification of histone post-translational modifications on a q exactive hf hybrid quadrupole orbitrap mass spectrometer.histone post-translational modifications (ptms) play key roles in regulating eukaryotic gene expression. mass spectrometry (ms) has emerged as a powerful method to characterize and quantify histone ptms as it allows unbiased identification and quantification of multiple histone ptms including combinations of the modifications present.201930701600
an oral toxicity test in rats and a genotoxicity study of extracts from the stems of opuntia ficus-indica var. saboten.opuntia ficus-indica var. saboten (ofis) is used widely in korea to treat constipation due to its diuretic effects and its enhancement of bowel function and appetite. however, its safety has not yet been established. the aim of this study was to evaluate the repeated oral toxicity and genotoxicity of ofis extract (oe).201930691445
silk sericin hydrolysate is a potential candidate as a serum-substitute in the culture of chinese hamster ovary and henrietta lacks cells.the silk sericin hydrolysate (ssh) from the waste of silk processing as a substitute of fetal bovine serum (fbs) was used for the culture of chinese hamster ovary (cho) cells and henrietta lacks (hela) strain of human cervical cancer cells. the survival ratio of these cells cultured in ssh media were similar to or higher than those in fbs media. especially after the serum was replaced by low concentration of ssh at 15.0 μg/ml for 5 d, the proliferation of both cells was also similar to or higher ...201930690536
current state-of-the-art in plant-based antibody production systems.monoclonal antibodies represent the major class of biopharmaceutical products (for therapeutics and diagnostics) with an increasing demand that reaches several tons per year worldwide. traditional large-scale manufacturing processes are based on stirred tank bioreactors for the growth of chinese hamster ovary cells (cho) which requires high initial investments and production costs. therefore, there is an urgent need for alternative production platforms that can at least act as a complement to th ...201930684155
dissecting n-glycosylation dynamics in chinese hamster ovary cells fed-batch cultures using time course omics analyses.n-linked glycosylation affects the potency, safety, immunogenicity, and pharmacokinetic clearance of several therapeutic proteins including monoclonal antibodies. a robust control strategy is needed to dial in appropriate glycosylation profile during the course of cell culture processes accurately. however, n-glycosylation dynamics remains insufficiently understood owing to the lack of integrative analyses of factors that influence the dynamics, including sugar nucleotide donors, glycosyltransfe ...201930682623
pmab-210: a monoclonal antibody against pig podoplanin.podoplanin (pdpn) is a type i transmembrane glycoprotein that is expressed in normal tissues, including renal corpuscles and type i lung alveolar cells. monoclonal antibodies (mabs) against human, mouse, rat, rabbit, dog, cat, and bovine pdpns have already been established; however, antipig pdpn (ppdpn) mabs have not. we therefore immunized mice with ppdpn-overexpressing chinese hamster ovary (cho)-k1 cells (cho/ppdpn), and screened hybridomas, which are producing anti-ppdpn mabs. one of mabs, p ...201930681406
multiple mechanisms underlie increased cardiac late sodium current.we recently reported a quantitative relationship between the degree of functional perturbation reported in the literature for 356 variants in the cardiac sodium channel gene scn5a and the penetrance of resulting arrhythmia phenotypes. in the course of that work, we identified multiple scn5a variants, including r1193q, that are common in populations but are reported in human embryonic kidney (hek) cells to generate large late sodium current (ina-l).201930677491
histidine-rich cell-penetrating peptide for cancer drug delivery and its uptake mechanism.in this work, we report a drug delivery system based on the ph-responsive self-assembly and -disassembly behaviors of peptides. here, a systematically designed histidine-rich lipidated peptide (np1) is presented to encapsulate and deliver an anticancer drug ellipticine (ept) into two model cells: non-small-cell lung carcinoma and chinese hamster ovary cells. the mechanism of ph-responsive peptide self-assembly and -disassembly involved in the drug encapsulation and release process are extensivel ...201930673275
chromophore pre-maturation for improved speed and sensitivity of split-gfp monitoring of protein secretion.complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. a split-gfp variant, involving the self-associating gfp 1-10 and gfp 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. a limitation of this approach is the slow chromophore formation after complementation. here, we alleviate this lag in signal generation by allowing the gfp 1-10 chromophore to mature on a solid support ...201930670736
generation of site-distinct n-glycan variants for in vitro bioactivity testing.glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a fc-fusion antibody comprizing three different n-glycosylation sites. by adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse n-glycan variant population was generated, including high mannose, afucosylated, fucosylated, aga ...201930659587
bmn 250, a fusion of lysosomal alpha-n-acetylglucosaminidase with igf2, exhibits different patterns of cellular uptake into critical cell types of sanfilippo syndrome b disease pathogenesis.sanfilippo syndrome type b (sanfilippo b; mucopolysaccharidosis type iiib) occurs due to genetic deficiency of lysosomal alpha-n-acetylglucosaminidase (naglu) and subsequent lysosomal accumulation of heparan sulfate (hs), which coincides with devastating neurodegenerative disease. because naglu expressed in chinese hamster ovary cells is not mannose-6-phosphorylated, we developed an insulin-like growth factor 2 (igf2)-tagged naglu molecule (bmn 250; tralesinidase alfa) that binds avidly to the i ...201930657762
use of a branched linker for enhanced biosensing properties in igg detection from mixed chinese hamster ovary cell cultures.tris(2-aminoethyl)-amine (tren), a branched amine, was coupled to planar surfaces of alkanethiol self-assembled monolayers (sams) to increase the grafting density of igg-binding peptide (hwrgwv or hwrgwvg) on gold surfaces. one of the three primary amine pendant groups of tren anchors onto the sam, while the other two are available for grafting with the c-termini of the peptide. the ellipsometric peptide density on the sam-branched amine was 1.24 molecules nm-2. the surfaces carrying the peptide ...201930653289
a novel methodology to assess linear energy transfer and relative biological effectiveness in proton therapy using pairs of differently doped thermoluminescent detectors.a new methodology for assessing linear energy transfer (let) and relative biological effectiveness (rbe) in proton therapy beams using thermoluminescent detectors is presented. the method is based on the different let response of two different lithium fluoride thermoluminescent detectors (lif:mg,ti and lif:mg,cu,p) for measuring charged particles. the relative efficiency of the two detector types was predicted using the recently developed microdosimetric d(z) model in combination with the monte ...201930650402
silkworm storage protein 1 inhibits autophagy-mediated apoptosis.autophagy is a natural physiological process, and it induces the lysosomal degradation of intracellular components in response to environmental stresses, including nutrient starvation. although an adequate autophagy level helps in cell survival, excessive autophagy triggered by stress such as starvation leads to autophagy-mediated apoptosis. chinese hamster ovary (cho) cells are widely used for producing biopharmaceuticals, including monoclonal antibodies. however, apoptosis induced by high stre ...201930646576
increased growth rate and productivity following stable depletion of mir-7 in a mab producing cho cell line causes an increase in proteins associated with the akt pathway and ribosome biogenesis.cell line engineering using micrornas represents a desirable route for improving the efficiency of recombinant protein production by cho cells. in this study we generated stable cho dp12 cells expressing a mir-7 sponge transcript which sequesters mir-7 from its endogenous targets. depletion of mir-7 results in a 65% increase in cell growth and >3-fold increase in yield of secreted igg protein. quantitative labelfree lc-ms/ms proteomic profiling was carried out to identify the targets of mir-7 an ...201930641232
secretion analysis of intracellular "difficult-to-express" immunoglobulin g (igg) in chinese hamster ovary (cho) cells.the chinese hamster ovary (cho) cell line is the most widely used host cell for therapeutic antibody production. although its productivity has been improved by various strategies to satisfy the growing global demand, some difficult-to-express (dte) antibodies remain at low secretion levels. to improve the production of various therapeutic antibodies, it is necessary to determine possible rate-limiting steps in dte antibody secretion in comparison with other high igg producers. here, we analyzed ...201930637508
analysis of golgi ph in chinese hamster ovary cells using ratiometric ph-sensitive fluorescent proteins.acidic golgi ph plays an important role in protein glycosylation, one of the critical quality attributes of therapeutic proteins. to determine the intracellular golgi ph during culture, stable chinese hamster ovary (cho) cell clones expressing phluorin2, a ratiometric ph-sensitive fluorescent protein (fp), in the cis- and trans-golgi, were constructed by fusing phluorin2 with specific targeting proteins, acetylglucosaminyltransferase, and a galactosyltransferase, respectively. stable cho cell cl ...201930636290
optimization of sequence, display, and mode of operation of igg-binding peptide ligands to develop robust, high-capacity affinity adsorbents that afford high igg product quality.this work presents the use of peptide ligand hwrgwv and its cognate sequences to develop affinity adsorbents that compete with protein a in terms of binding capacity and quality of the eluted product. first, the peptide ligand was conjugated to crosslinked agarose resins (workbeads) at different densities and using different spacer arms. the optimization of ligand density and display resulted in values of static and dynamic binding capacity of 85 mg/ml and 65 mg/ml, respectively. a selected pept ...201930621158
zinc oxide nanoparticles induced gene mutation at the hgprt locus and cell cycle arrest associated with apoptosis in v-79 cells.in recent years, the large-scale production of zno nanoparticles (nps) for various applications is increasing exponentially and may pose serious health issues when inhaled either during occupational exposure or in consumer settings. the mechanisms underlying the toxicity of nps have recently been studied intensively. despite the existing studies, the mutagenicity of zno nps in the eukaryotic system is still unclear. therefore, the aim of the present study was to investigate the mutagenic potenti ...201930618096
expression in eukaryotic cells and purification of synthetic gene encoding enterocin p: a bacteriocin with broad antimicrobial spectrum.due to the emergence of multidrug-resistant bacteria, treatment options for infectious diseases are decreasing. bacteriocins are small antimicrobial peptides produced by numerous bacteria that offer alternative therapeutic strategies to combat multidrug-resistant bacterial infections. we evaluated the cloning, functional expression, and antimicrobial activities of enterocin p (entp), a class ii bacteriocin member, in chinese hamster ovary (cho) cells. a synthetic gene matching cho cell codon usa ...201930617751
anti-hyperglycemic and genotoxic studies of 1-o-methyl chrysophanol, a new anthraquinone isolated from amycolatopsis thermoflava strain sfma-103.the compound 1-o-methyl chrysophanol (omc) which belongs to a class of hydroxyanthraquinones was isolated from amycolatopsis thermoflava strain sfma-103 and studied for their anti-diabetic properties. omc was evaluated as an anti-diabetic agent based on in silico studies which initially predicted the binding energy with α-amylase (-188.81 kj mol-1) and with α-glucosidase (70.53 kj mol-1). further, these results were validated based on enzyme inhibition assays where omc demonstrated enzyme inhibi ...201930614298
analysis of the immunoglobulin g (igg) secretion efficiency in recombinant chinese hamster ovary (cho) cells by using citrine-fusion igg.biopharmaceuticals represented by immunoglobulin g (igg) are produced by the cultivation of recombinant animal cells, especially chinese hamster ovary (cho) cells. it is thought that the intracellular secretion process of igg is a bottleneck in the production of biopharmaceuticals. many studies on the regulation of endogenous secretory protein expression levels have shown improved productivity. however, these strategies have not universally improved the productivity of various proteins. a more r ...201930610509
comparison of integrases identifies bxb1-ga mutant as the most efficient site-specific integrase system in mammalian cells.phage-derived integrases can catalyze irreversible, site-specific integration of transgenic payloads into a chromosomal locus, resulting in mammalian cells that stably express transgenes or circuits of interest. previous studies have demonstrated high-efficiency integration by the bxb1 integrase in mammalian cells. here, we show that a point mutation (bxb1-ga) in bxb1 target sites significantly increases bxb1-mediated integration efficiency at the rosa26 locus in chinese hamster ovary cells, res ...201930609349
a pathogenic tau fragment compromises microtubules, disrupts insulin signaling and induces the unfolded protein response.human tauopathies including alzheimer's disease, progressive supranuclear palsy and related disorders, are characterized by deposition of pathological forms of tau, synaptic dysfunction and neuronal loss. we have previously identified a pathogenic c-terminal tau fragment (tau35) that is associated with human tauopathy. however, it is not known how tau fragmentation affects critical molecular processes in cells and contributes to impaired physiological function. chinese hamster ovary (cho) cells ...201930606258
fmoc-ff and hexapeptide-based multicomponent hydrogels as scaffold materials.short peptides or single amino acids are interesting building blocks for fabrication of hydrogels, frequently used as extracellular matrix-mimicking scaffolds for cell growth in tissue engineering. the combination of two or more peptide hydrogelators could allow obtaining different materials exhibiting new architectures, tunable mechanical properties, high stability and improved biofunctionality. here we report on the synthesis, formulation and multi-scale characterization of peptide-based mixed ...201930601569
interaction of cell culture process parameters for modulating mab afucosylation.the extent of afucosylation, which refers to the absence of core fucose on fc glycans, can correlate positively with the antibody-dependent cellular cytotoxicity (adcc) activity of a monoclonal antibody (mab). therefore, it is important to maintain consistent afucosylation during cell culture process scale-up in bioreactors for a mab with adcc activity. however, there is currently a lack of understanding about the impact of partial pressure of carbon dioxide (pco2 )-a parameter that can vary wit ...201930597531
acute and genetic toxicity of gs-e3d, a new pectin lyase-modified red ginseng extract.korean red ginseng and its extract have been used as traditional medicines and functional foods in countries worldwide. pectin lyase-modified red ginseng extract (gs-e3d) was newly developed as a dietary supplement for obesity, diabetes-related renal dysfunction, etc. in this study, the safety of gs-e3d on acute toxicity and genotoxicity was evaluated. for acute study, sprague-dawley rats were administrated by oral gavage at a dose of 5000 mg/kg gs-e3d. to evaluate genotoxicity of gs-e3d, we con ...201930904430
identification of mammalian glycoproteins with type-i lacdinac structures synthesized by the glycosyltransferase b3galnt2.the type-i lacdinac (ldn; galnacβ1-3glcnac) has rarely been observed in mammalian cells except in the o-glycan of α-dystroglycan, in contrast to type-ii ldn structures (galnacβ1-4glcnac) in n- and o-glycans that are present in many mammalian glycoproteins, such as pituitary and hypothalamic hormones. although a β1,3-n-acetylgalactosaminyltransferase 2 (b3galnt2; type-i ldn synthase) has been cloned, the function of type-i ldn in mammalian cells is still unclear, as its carrier protein(s) has not ...201930898876
acaricidal mechanism of scopoletin against tetranychus cinnabarinus.scopoletin is a promising acaricidal botanical natural compound against tetranychus cinnabarinus, and its acaricidal mechanism maybe involve calcium overload according to our previous study. to seek potential candidate target genes of calcium overload induced by scopoletin in t. cinnabarinus, rna-seq was utilized to detect changes in transcription levels. 24 and 48 h after treatment, 70 and 102 differentially expressed genes were obtained, respectively. target genes included 3 signal transductio ...201930894818
reducing recombinant protein expression during cho pool selection enhances frequency of high-producing cells.chinese hamster ovary (cho) cells are the most widely used mammalian host for industrial-scale production of monoclonal antibodies (mabs) and other protein biologics. isolation of rare high-producing cho cell lines from heterogeneous populations of stable transfectants is a daunting task and delays the process of manufacturing of novel biologics. a variety of factors that contribute to the low frequency of high-producing clones have been described; however, the impact of metabolic burden and oth ...201930885656
cytoskeletal synchronization of cho cells with polymer functionalized fullerene c60.recent developments in the field of fullerene c60 and its derivatives suggest its suitability in a wide range of applications ranging from photovoltaic instruments, development of solar based cells, cosmetics to enzyme inhibition treatment, and so on. these innovative applications raised possibilities of intentional or oblivious human-particle contact leading to possible deleterious effects on human health. the current study deals with the interaction of dextran functionalized fullerene c60 (dex ...201930884950
the effects of pertuzumab and its combination with trastuzumab on her2 homodimerization and phosphorylation.pertuzumab (perjeta) is an anti-her2 monoclonal antibody that is used for treatment of her2-positive breast cancers in combination with trastuzumab (herceptin) and docetaxel and showed promising clinical outcomes. pertuzumab is suggested to block heterodimerization of her2 with egfr and her3 that abolishes canonical function of her2. however, evidence on the exact mode of action of pertuzumab in homodimerization of her2 are limited. in this study, we investigated the effect of pertuzumab and its ...201930884851
reduced apoptosis in chinese hamster ovary cells via optimized crispr interference.chinese hamster ovary (cho) cells are widely used for biopharmaceutical protein production. one challenge limiting cho cell productivity is apoptosis stemming from cellular stress during protein production. here we applied crispr interference (crispri) to downregulate the endogenous expression of apoptotic genes bak, bax, and casp3 in cho cells. in addition to reduced apoptosis, mitochondrial membrane integrity was improved and the caspase activity was reduced. moreover, we optimized the crispri ...201930883679
impact of depth filtration on disulfide bond reduction during downstream processing of monoclonal antibodies from cho cell cultures.monoclonal antibody interchain disulfide bond reduction was observed in a chinese hamster ovary manufacturing process that used single-use technologies. a similar reduction has been reported for processes that involved high mechanical shear recovery unit operations, such as continuous flow centrifugation and when the clarified harvest was stored under low dissolved oxygen (do) conditions (trexler-schmidt et al., 2010. biotechnology and bioengineering, 106(3), 452-461). the work described here id ...201930883673
carbosilane dendrimers with phosphonium terminal groups are low toxic non-viral transfection vectors for sirna cell delivery.non-viral gene delivery vectors studied in the gene therapy applications are often designed with the cationic nitrogen containing groups necessary for binding and cell release of nucleic acids. disadvantage is a relatively high toxicity which restricts the in vivo use of such nanoparticles. here we show, that the 3rd generation carbosilane dendrimers possessing (trimethyl)phosphonium (pme3) groups on their periphery were able to effectively deliver the functional sirna into the cells (b14, crice ...201930877030
progression of change in membrane capacitance and cytoplasm conductivity of cells during controlled starvation using dual-frequency dep cytometry.the dielectric properties of cells are directly related to their morphological and physiological properties and can be used to monitor their status when exposed to stress conditions. in this work, dual-frequency dielectrophoresis (dep) cytometry was employed to measure changes in the membrane capacitance and cytoplasm conductivity of single chinese hamster ovary (cho) cells during the progression of starvation-induced apoptosis. our dual-frequency dep cytometer enables simultaneous measurement o ...201930876633
the effect of green and black tea polyphenols on brca2 deficient chinese hamster cells by synthetic lethality through parp inhibition.tea polyphenols are known antioxidants presenting health benefits due to their observed cellular activities. in this study, two tea polyphenols, epigallocatechin gallate, which is common in green tea, and theaflavin, which is common in black tea, were investigated for their parp inhibitory activity and selective cytotoxicity to brca2 mutated cells. the observed cytotoxicity of these polyphenols to brca2 deficient cells is believed to be a result of parp inhibition induced synthetic lethality. ch ...201930875717
virus removal filtration of chemically defined chinese hamster ovary cells medium with nanocellulose-based size exclusion filter.sterility of bioreactors in biotherapeutic processing remains a significant challenge. virus removal size-exclusion filtration is a robust and highly efficient approach to remove viruses. this article investigates the virus removal capacity of nanocellulose-based filter for upstream bioprocessing of chemically defined chinese hamster ovary (cho) cells medium containing pluronic f-68 (powercho™, lonza) and supplemented with insulin-transferrin-selenium (its) at varying process parameters. virus r ...201930871931
host-antibody inductivity of virulent entamoeba histolytica and non-virulent entamoeba moshkovskii in a mouse model.despite similarities in morphology, gene and protein profiles, entamoeba histolytica and e. moshkovskii show profound differences in pathogenicity. entamoeba histolytica infection might result in amoebic dysentery and liver abscess, while e. moshkovskii causes only mild diarrhea. extensive studies focus on roles of host immune responses to the pathogenic e. histolytica; however, evidence for e. moshkovskii remains scarce.201930867040
a novel led-based 2d-fluorescence spectroscopy system for in-line bioprocess monitoring of chinese hamster ovary cell cultivations-part ii.this study was performed in order to evaluate a new led-based 2d-fluorescence spectrometer for in-line bioprocess monitoring of chinese hamster ovary (cho) cell culture processes. the new spectrometer used selected excitation wavelengths of 280, 365, and 455 nm to collect spectral data from six 10-l fed-batch processes. the technique provides data on various fluorescent compounds from the cultivation medium as well as from cell metabolism. in addition, scattered light offers information about th ...201932625013
both irradiated and bystander effects link with dna repair capacity and the linear energy transfer.in comparison with a low linear energy transfer (let) radiation, a high-let radiation induces more complex dna damage. this study wonders whether radiation-induced bystander effect (ribe) is dependent of let.201930858123
selection of vaccinia virus-neutralizing antibody from a phage-display human-antibody library.although smallpox was eradicated in 1980, it is still considered a potential agent of biowarfare and bioterrorism. smallpox has the potential for high mortality rates along with a major public health impact, eventually causing public panic and social disruption. passive administration of neutralizing monoclonal antibodies (mabs) is an effective intervention for various adverse reactions caused by vaccination and the unpredictable nature of emerging and bioterrorist-related infections. currently, ...201930856707
secreting oviduct epithelial cells of coturnix coturnix japonica (qoec) and changes to their proteome after nonviral transfection.the quail oviduct (coturnix c. japonica) is a natural candidate avian bioreactor, while the secretive quail oviduct epithelial cells (qoecs) are potential in vitro producers of recombinant proteins and vaccines. in view of the need for highly performing and transformable cell lines, qoec may potentially act as an alternative bioreactor platform to the existing ones, for example, to the chinese hamster ovary. the aim of this work was to characterize qoecs and their response to nucleofection with ...201930854717
transient manipulation of the expression level of selected growth rate correlating micrornas does not increase growth rate in cho-k1 cells.engineering of chinese hamster ovary cells by manipulating microrna (mirna) expression levels has been shown to induce advantageous, desired phenotypes. most of these studies so far were concerned with increasing productivity or reducing growth rate (with the implied intention of thus freeing cellular resources to also increase productivity). here we evaluated the ability of growth correlating mirnas to increase the growth rate of cho-k1 cells by transient overexpression or knock down, respectiv ...201930853633
rapid establishment of stable retroviral packaging cells and recombinant susceptible target cell lines employing novel transposon vectors derived from sleeping beauty.viral vector particles derived from murine leukemia virus (mlv) mediate highly efficient stable gene transfer used in gene therapeutic approaches and in the generation of transgenic cell lines. however, the establishment of stable viral packaging cells (vpcs) is a time-consuming challenge. to overcome this limitation, we successfully generated novel sleeping beauty-derived transposon vectors entailing envelope and packaging expression cassettes as well as a transfer vector. upon multiplexed tran ...201930852270
metabolic engineering of chinese hamster ovary cells towards reduced biosynthesis and accumulation of novel growth inhibitors in fed-batch cultures.chinese hamster ovary (cho) cells in fed-batch cultures are known to consume large amounts of nutrients and divert significant portion of them towards the formation of byproducts, some of which, including lactate and ammonia, are known to be growth inhibitory in nature. a major fraction of these inhibitory metabolites are byproducts or intermediates of amino acid catabolism. limiting the supply of amino acids has been shown to curtail the production of corresponding inhibitory byproducts resulti ...201930851381
chemical, cytotoxic and genotoxic analysis of etidronate in sodium hypochlorite solution.to test whether the incorporation of a chelation powder, etidronate, marketed for root canal irrigation (dual rinse hedp) into a sodium hypochlorite (naocl) solution induced additional cytotoxic and genotoxic effects not observed with naocl alone.201930848496
in vitro and in vivo evaluation of 6-o-α-maltosyl-β-cyclodextrin as a potential therapeutic agent against niemann-pick disease type c.niemann-pick disease type c (npc) is a rare lysosomal storage disease characterized by the dysfunction of intracellular cholesterol trafficking with progressive neurodegeneration and hepatomegaly. we evaluated the potential of 6-o-α-maltosyl-β-cyclodextrin (g2-β-cd) as a drug candidate against npc. the physicochemical properties of g2-β-cd as an injectable agent were assessed, and molecular interactions between g2-β-cd and free cholesterol were studied by solubility analysis and two-dimensional ...201930845767
crispr/cas9 targeted capture of mammalian genomic regions for characterization by ngs.the robust detection of structural variants in mammalian genomes remains a challenge. it is particularly difficult in the case of genetically unstable chinese hamster ovary (cho) cell lines with only draft genome assemblies available. we explore the potential of the crispr/cas9 system for the targeted capture of genomic loci containing integrated vectors in cho-k1-based cell lines followed by next generation sequencing (ngs), and compare it to popular target-enrichment sequencing methods and to ...201930837529
induction of peripheral lymph node addressin in human nasal mucosa with eosinophilic chronic rhinosinusitis.eosinophilic chronic rhinosinusitis (ecrs) is characterised by formation of nasal polyps with prominent eosinophilic infiltration; however, how eosinophils are recruited in this pathological setting remains unclear. in the present study, we carried out quantitative immunohistochemical analysis of nasal polyps associated with ecrs (n=30) and non-ecrs (n=30) to evaluate expression of an l-selectin ligand peripheral lymph node addressin (pnad) on vascular endothelial cells. we found that pnad was i ...201930837082
site-specific roles of n-linked oligosaccharides in recombinant eel follicle-stimulating hormone for secretion and signal transduction.eel follicle-stimulating hormone (eelfsh) is composed of a common α-subunit and a hormone specific β-subunit, both of which contain two n-linked carbohydrate residues. we characterized the biologically active single chains by fusing the α-subunit to the carboxyl terminal region of the eelfsh β-subunit. expression vectors were constructed and the biological activity of the recombinant hormones (rec-hormones) was characterized using chinese hamster ovary (cho) k1 cells expressing the eelfsh recept ...201930836102
electrophysiological and pharmacological characterization of human inwardly rectifying kir2.1 channels on an automated patch-clamp platform.inwardly rectifying ik1 potassium currents of the heart control the resting membrane potential of ventricular cardiomyocytes during diastole and contribute to their repolarization after each action potential. mutations in the gene encoding kir2.1 channels, which primarily conduct ventricular ik1, are associated with inheritable forms of arrhythmias and sudden cardiac death. therefore, potential iatrogenic inhibition of kir2.1-mediated ik1 currents is a cardiosafety concern during new drug discov ...201930835490
evaluation of pertussis toxin expression in b2 and thijs media.whole-cell pertussis vaccine (wp) has been imperative and highly effective in preventing childhood deaths due to pertussis. pertussis toxin is one of the virulence factors of bordetella pertussis in all available pertussis vaccines. wp production in razi vaccine and serum research institute is according to bioreactor culture of b. pertussis strains in b2 medium. the aim of this study was to evaluate b. pertussis strain 509 pt production in b2 and thalen-ijssel (thijs) media by chinese hamster ov ...201931013007
establishment of a monoclonal antibody pmab-225 against alpaca podoplanin for immunohistochemical analyses.podoplanin (pdpn) is known as a lymphatic endothelial cell marker. monoclonal antibodies (mabs) against human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse pdpn have been established in our previous studies. however, mabs against alpaca pdpn (apdpn), required for immunohistochemical analysis, remain to be developed. in the present study, we employed the cell-based immunization and screening (cbis) method for producing anti-apdpn mabs. we immunized mice with apdpn-overexpressing chinese h ...201930997422
predicting the cytotoxicity of disinfection by-products to chinese hamster ovary by using linear quantitative structure-activity relationship models.a suitable model to predict the toxicity of current and continuously emerging disinfection by-products (dbps) is needed. this study aims to establish a reliable model for predicting the cytotoxicity of dbps to chinese hamster ovary (cho) cells. we collected the cho cytotoxicity data of 74 dbps as the endpoint to build linear quantitative structure-activity relationship (qsar) models. the linear models were developed by using multiple linear regression (mlr). the mlr models showed high performanc ...201930989598
development of a long-acting fc-fused cocaine hydrolase with improved yield of protein expression.human butyrylcholinesterase (bche) is known as a safe and effective protein for detoxification of organophosphorus (op) nerve agents. its rationally designed mutants with considerably improved catalytic activity against cocaine, known as cocaine hydrolases (cochs), are recognized as the most promising drug candidates for the treatment of cocaine abuse. however, it is a grand challenge to efficiently produce active recombinant bche and cochs with a sufficiently long biological half-life. in the p ...201930986387
application of the bradford assay for cell lysis quantification: residual protein content in cell culture supernatants.frequently measured mammalian cell culture process indicators include viability and total cell concentration (tcc). cell lysis, an additional important process characteristic that substantially contributes to the overall product purity profiles, is often not addressed in detail. in the present study, an inexpensive and simple application of the bradford assay is developed to determine the residual protein content (rpc) in cell culture supernatants. the reliability and reproducibility of the meth ...201930983130
three consecutive pregnancies in a patient with chronic autoimmune thyroid disease associated with hypothyroidism and extremely high levels of anti-thyrotropin receptor antibodies.background: thyroid-stimulating hormone (tsh) receptor (tshr) antibodies (trab) can be present in chronic autoimmune thyroiditis. transplacental trab transfer can lead to fetal thyroid dysfunction and serious complications. patient findings: we report the case of a woman with autoimmune hypothyroidism and extremely high trab levels, with blocking and stimulating activities (biological activities characterized with chinese hamster ovary cells expressing tshr). at week 22 of her first pregnancy, s ...201930973063
anxa2- and ctsd-knockout cho cell lines to diminish the risk of contamination with host cell proteins.chinese hamster ovary (cho) cells have been used as host cells in the production of a range of recombinant therapeutic proteins, including monoclonal antibodies and fc-fusion proteins. host cell proteins (hcp) represent impurities that must be removed from therapeutic formulations because of their potential risks for immunogenicity. while the majority of hcp impurities are effectively removed in typical downstream purification processes, clearance of a small population of hcp remains challenging ...201930972970
a combined cell-free protein synthesis and fluorescence-based approach to investigate gpcr binding properties.fluorescent labeling of de novo synthesized proteins is in particular a valuable tool for functional and structural studies of membrane proteins. in this context, we present two methods for the site-specific fluorescent labeling of difficult-to-express membrane proteins in combination with cell-free protein synthesis. the cell-free protein synthesis system is based on chinese hamster ovary cells (cho) since this system contains endogenous membrane structures derived from the endoplasmic reticulu ...201930969411
genetic and epigenetic variation across genes involved in energy metabolism and mitochondria of chinese hamster ovary cell lines.the increasingdemandfor biopharmaceutical products drives the search for efficient cell factories that are able to sustainably support rapid growth, high productivity, and product quality. as these depend on energy generation, here the genomic variation in nuclear genes associated with mitochondria and energy metabolism and the mitochondrial genome of 14 cell lines is investigated. the variants called enable reliable tracing of lineages. unique sequence variations are observed in cell lines adap ...201930969017
targeted capture of chinese hamster ovary host cell proteins: peptide ligand discovery.the growing integration of quality-by-design (qbd) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. particularly valuable is the determination of the residual level of host cell proteins (hcps) secreted, together with the product of interest, by the recombinant cells utilized for production. though often referred to as a single impurity, hcps comprise a variety of species with diverse ab ...201930965558
hydrophobic amines and their guanidine analogues modulate activation and desensitization of asic3.acid-sensing ion channel 3 (asic3) is an important member of the acid-sensing ion channels family, which is widely expressed in the peripheral nervous system and contributes to pain sensation. asics are targeted by various drugs and toxins. however, mechanisms and structural determinants of ligands' action on asic3 are not completely understood. in the present work we studied asic3 modulation by a series of "hydrophobic monoamines" and their guanidine analogs, which were previously characterized ...201930959896
identification of consensus sequence from matrix attachment regions and functional analysis of its activity in stably transfected chinese hamster ovary cells.matrix attachment regions (mars) can enhance transgene expression levels and maintain stability. however, the consensus sequence from mars and its functional analysis remains to be examined. here, we assessed a possible consensus sequence from mars and assessed its activity in stably transfected chinese hamster ovary (cho) cells. first, we analyzed the effects of 10 mars on transfected cho cells and then analyzed the consensus motifs from these mars using a bioinformatics method. the consensus s ...201930957285
human genome-derived top1 matrix attachment region enhances transgene expression in the transfected cho cells.to investigate the effect of full-length fragment of dna topoisomerase i gene (top1) matrix attachment regions (mars) originating from the human genome on transgene expression in chinese hamster ovary (cho) cells and explore the underlying mechanisms.201930953310
a novel led-based 2d-fluorescence spectroscopy system for in-line monitoring of chinese hamster ovary cell cultivations - part i.a new two-dimensional fluorescence sensor system was developed for in-line monitoring of mammalian cell cultures. fluorescence spectroscopy allows for the detection and quantification of naturally occurring intra- and extracellular fluorophores in the cell broth. the fluorescence signals correlate to the cells' current redox state and other relevant process parameters. cell culture pretests with twelve different excitation wavelengths showed that only three wavelengths account for a vast majorit ...201932625014
use of virus-like particles as a native membrane model to study the interaction of insulin with the insulin receptor.there is emerging evidence of the utility of virus-like particles (vlps) as a novel model for the study of receptor-ligand interactions in a native plasma membrane environment. vlps consist of a viral core protein encapsulated by portions of the cell membrane with membrane proteins and receptors expressed in their native conformation. vlps can be generated in mammalian cells by transfection with the retroviral core protein (gag). in this study, we used chinese hamster ovary (cho t10) cells stabl ...201930951702
nanodiamond/poly-ε-caprolactone nanofibrous scaffold for wound management.we describe preparation, characterization and cytocompatibility of nanodiamond (nd) dispersed in poly (ε-caprolactone) (pcl) based nanofibrous scaffold. the results show that this unique scaffold potentially provides essential properties for wound healing by enhancing proliferation of epithelial cells, in addition to restricting the microbial activities. electrospinning technique was used to fabricate and develop pcl-nds nanocomposite scaffold. the developed nanocomposites were characterized for ...201930948073
escherichia coli bmp-2 showed comparable osteoinductivity with chinese hamster ovary derived bmp-2 with demineralized bone matrix as carrier.escherichia coli bone morphogenetic protein-2 (erhbmp-2) had a larger yield but less osteoinductivity than chinese hamster ovary cell bone morphogenetic protein-2 (crhbmp-2). since the release profile of rhbmp-2 affects its osteoinductivity, an appropriate carrier could improve the effect of erhbmp-2. demineralized bone matrix (dbm) was one of the most widely used bone substitutes, but few studies evaluated the osteoinductivity of erhbmp-2 while it was carried by dbm. therefore, we compared the ...201930947586
exploring isoxazoles and pyrrolidinones decorated with the 4,6-dimethoxy-1,3,5-triazine unit as human farnesyltransferase inhibitors.unprecedented triazinyl-isoxazoles were afforded via an effective cycloaddition reaction between nitrile oxides and the scarcely described 2-ethynyl-4,6-dimethoxy-1,3,5-triazine as dipolarophile. the biological evaluation of the newly synthesized compounds showed that the inhibition of human farnesyltransferase by zinc complexation could be improved with triazine-isoxazole moieties. the replacement of the isoxazole unit by a pyrrolidin-2-one was detrimental to the inhibitory activity while the p ...201930947375
risk assessment of heterogeneous tio2-based engineered nanoparticles (nps): a qstr approach using simple periodic table based descriptors.nowadays, the risk assessment of engineered nanoparticles (nps) on human health and animals is of great importance. we have used here simple periodic table based descriptors for mixture compounds to predict the cytotoxicity for the heterogeneous nps. we have developed mono parametric quantitative structure-toxicity relationship (qstr) models for 34 tio2-based nps modified with (poly) metallic clusters of noble metals (au, ag, pt) to assess the cytotoxicity (-log ec50) towards chinese hamster ova ...201930938199
revisiting the mechanism of enfuvirtide and designing an analog with improved fusion inhibitory activity by targeting triple sites in gp41.to revisit the mechanism of action of enfuvirtide (t20) and based on the newly defined mechanism, design an analogous peptide of t20 with improved antiviral activity.201930932963
from nutritional wealth to autophagy: in vivo metabolic dynamics in the cytosol, mitochondrion and shuttles of igg producing cho cells.to fulfil the optimization needs of current biopharmaceutical processes the knowledge how to improve cell specific productivities is of outmost importance. this requires a detailed understanding of cellular metabolism on a subcellular level inside compartments such as cytosol and mitochondrion. using igg1 producing chinese hamster ovary (cho) cells, a pioneering protocol for compartment-specific metabolome analysis was applied. various production-like growth conditions ranging from ample glucose ...201930930288
recombinant human erythropoietin proteins synthesized in escherichia coli cells: effects of additional domains on the in vitro and in vivo activities.the aim of this work was to compare biological activities of three variants of bacterially expressed human recombinant erythropoietin (epo) with additional protein domains: 6his-s-tag-epo protein carrying the s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease a) at the n-terminus and hbd-epo and epo-hbd proteins containing heparin-binding protein domains (hbd) of the bone morphogenetic protein 2 from danio rerio at the n- and c-termini, respectively. the commercial preparation epost ...201930927522
application of imaging flow cytometry for the characterization of intracellular attributes in chinese hamster ovary cell lines at the single-cell level.biopharmaceutical manufacturing using chinese hamster ovary (cho) cells requires the generation of high-producing clonal cell lines. during cell line development, cell cloning using fluorescence-activated cell sorting (facs) has the potential to combine isolation of single cells with sorting based on specific cellular attributes that correlate with productivity and/or growth, identifying cell lines with desirable phenotypes for manufacturing. this study describes the application of imaging flow ...201930925020
simultaneous detection of nicotinamide adenine nucleotides and adenylate pool to quantify redox and energy states in mab-producing cho cells by capillary electrophoresis.chinese hamster ovary (cho) cells are predominant in the production of therapeutic proteins to treat various diseases. characterization and investigation of cho cell metabolism in a quick and simple way could boost process and cell line development. therefore, a method to simultaneously detect seven redox- and energy-related metabolites in cho cells by capillary electrophoresis has been developed. an on-line focusing technique was applied to improve the peak shape and resolution by using a 50 μm ...201930923861
crispr/cas9 as a genome editing tool for targeted gene integration in cho cells.the emergence of crispr/cas9 system as a precise and affordable method for genome editing has prompted its rapid adoption for the targeted integration of transgenes in chinese hamster ovary (cho) cells. targeted gene integration allows the generation of stable cell lines with a controlled and predictable behavior, which is an important feature for the rational design of cell factories aimed at the large-scale production of recombinant proteins. here we present the protocol for crispr/cas9-mediat ...201930912048
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