Dissertations / Theses on the topic 'Chinese Hamster Ovary cells'
To see the other types of publications on this topic, follow the link: Chinese Hamster Ovary cells.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Chinese Hamster Ovary cells.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Syddall, Katie Louise. "Directed evolution of Chinese Hamster Ovary cells." Thesis, University of Sheffield, 2016. http://etheses.whiterose.ac.uk/13836/.
Full text
2
Goh, Shireen. "Micro-bioreactor design for Chinese hamster ovary cells." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/82368.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2013.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 195-203).
The research objective is to design a micro-bioreactor for the culture of Chinese Hamster Ovary (CHO) cells. There is an increasing demand for upstream development in high-throughput micro-bioreactors specifically for the recombinant CHO cell line, an important cell line for producing recombinant protein therapeutics. In order to translate a micro-bioreactor originally designed by our group for bacteria to CHO cells, there would need to be significant modifications in the design of the micro-bioreactor due to the extreme sensitivity of CHO cells to physical and chemical stresses. Shear stresses inside the growth chamber will have to be reduced by three orders of magnitude. Moreover, the long doubling time of CHO cells requires a 2 weeks long culture. In a high surface to volume ratio micro-bioreactor, evaporation becomes a major problem. Contamination control is also vital for CHO cultures. In addition, the offline sampling volume required for validation necessitates a doubling of the working volume to 2mL. The newly designed Resistive Evaporation Compensated Actuated (RECA) micro-bioreactor is fully characterized in this thesis to ensure that the design meets the physical specifications of the required CHO cell culture conditions. The RECA micro-bioreactor will be tested with industrial recombinant CHO cell lines. This work is done in collaboration with Genzyme, USA and Sanofi-Aventis, Frankfurt. In this thesis, we also propose the use of dielectric spectroscopy electrodes for online cell viability sensing of CHO cells in micro-bioreactors. The electrodes are fabricated on polycarbonate, a biocompatible and optically clear thermoplastic that will be one of the future base material for microfluidic devices which can be rapidly prototyped. To demonstrate the viability of dielectric spectroscopy as an online viability sensor for CHO cells in a micro-bioreactor, the electrodes are used to characterize samples taken daily from a CHO shake flask batch culture without any sample modifications. Two different electrode geometries and correction methods will be compared to find the optimal system for viability measurements in a micro-bioreactor.
by Shireen Goh.
Ph.D.
3
Medvedeva, Natalia Gennadievna. "Influence of cell environment on micronucleation in Chinese hamster ovary cells." Texas A&M University, 2004. http://hdl.handle.net/1969.1/2790.
Full textAbstract:
The irradiation of cells in culture is an essential part of many radiation biology experiments. Since these experiments necessarily involve the irradiation of cell culture vessels and nutrient medium, the possibility of effects due to the interactions of irradiated material with growing cells needed to be investigated.
In the present study the micronucleus frequency in Chinese hamster ovary (CHO) cells as a function of such parameters as type of radiation, type of cell substrate, changes in cell environment, and time course of the effect were characterized. Observations of the persistence of micronucleus formation in irradiated CHO cells reveal that the number of cells containing micronuclei reaches its maximum within nine hours after irradiation and remain elevated for at least five days. The influence of the cell environment on micronucleus formation in CHO cells was examined by plating cells in preirradiated nutrient medium or on preirradiated cell culture vessels. In all experiments, pre-irradiation of the cell substrate (the culture dish or culture dish filled with medium) led to a significantly higher micronucleus frequency than when cells were plated on un-irradiated substrate. The difference is most pronounced at the lowest doses examined.
These results suggest that methods of cell culture vessel sterilization and the composition of cell attachment surfaces could be confounding factors, particularly in the experiments which are intended to examine the response of cells exposed to low doses of ionizing radiation.
4
Burford, Neil Thornton. "Cell signalling in Chinese hamster ovary cells expressing recombinant muscarinic receptors." Thesis, University of Leicester, 1994. http://hdl.handle.net/2381/33575.
Full textAbstract:
Agonist stimulation of recombinant m1, m2, and m3 muscarinic receptors, expressed in Chinese hamster ovary (CHO) cells, was compared. Carbachol binding affinity, and its modification by cations, guanine nucleotides and PTX pretreatment, was compared in washed membrane preparations of each of the CHO cell clones. Functional responses, determined by carbachol stimulation, were: [35S]GTPS binding in membranes; Ins(l,4,5)P3 accumulation in intact cells; 45ca2+ release from permeabilized cells; and cAMP accumulation in intact cells. m2-Transfected CHO cells were found to couple to AC, mediating inhibition of forskolin-stimulated cAMP accumulation, via PTX-sensitive G proteins. After PTX pretreatment of these cells, carbachol mediated a small potentiation of forskolin-stimulated cAMP accumulation, though with a much lower carbachol potency compared with the inhibitory response. m1 and m3-transfected CHO cell clones were found to couple with both PTX-sensitive and PTX-insensitive G proteins, at relatively high levels of receptor expression. The PTX- insensitive G proteins mediated agonist-stimulated PLC activation and were involved in the activation of AC (though at a much lower potency). The mechanism, by which m1, m2 and m3 muscarinic receptors stimulated AC activity was not thought to be due to crosstalk via PLC activity. The level of m3 muscarinic receptor expression, in CHO cells, was found to markedly affect both the potency, and the maximal responsiveness, with which carbachol mediated PLC activation and AC activation. Furthermore, at lower levels of receptor expression, m3 muscarinic receptors appeared to couple to a lesser extent with PTX-sensitive G proteins. The study, therefore, concluded that comparisons of agonist-mediated responses between muscarinic receptor subtypes, expressed in CHO cells, must be performed at similar levels of receptor expression. At similar receptor densities, m1 and m3 muscarinic receptors, in CHO cells, produced very similar responses to carbachol.
5
Thompson, Ben C. "Design of transient production systems with Chinese hamster ovary cells." Thesis, University of Sheffield, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.578708.
Full textAbstract:
Transient protein production by cultured mammalian cells from transfected episomal DNA is frequently used in bioindustry to generate small quantities of candidate therapeutic products during early stages of process development. However, transient production processes typically exhibit low productivity, limiting their use at scale. In this thesis, three distinct but complementary approaches were evaluated for the de novo design of a high productivity scaleable transient production process starting with the discrete raw materials: transfection reagent, Chinese hamster ovary cell line, plasmid DNA and chemically defined medium. (1) Optimisation of CHO host cell transfection: The optimal combination of continuous basal parameters underpinning polyethylenimine (PEI) mediated transfection (relative concentrations of PEI, plasmid DNA and cells) was determined utilising Design of Experiments (DoE) methodology. Optimum transfection conditions were cell line specific - highly dependent upon resistance to PEI cytotoxicity. Comparing different CHO cell hosts operating at their unique optima, variations in specific productivity were limited by the rate of polyplex endocytosis. (2) Modulation of the cell culture environment: Combinations of environmental variables were evaluated using factorial screening to determine an optimal cell culture regime for transient production. For the CHO cells used in this study, the addition of valproic acid, recombinant insulin-like growth factor and a reduced culture temperature were found to interact synergistically to maximise recombinant product yield at an increased cell concentration. (3) Production process design: Utilising response surface modelling to determine key process interactions, transient transfection and medium environment optima were effectively combined to create an intensified, high cell density process exhibiting a five-fold increase in volumetric titre. Combining these approaches, volumetric yield for a transient monoclonal antibody production process was increased from 2 mg L-1 to > 90 mg L-1 - the highest transient volumetric titre achieved with un-genetically modified CHO cells in a chemically defined environment to date.
6
Coppen, Steven Russell. "Studies on the aggregation of recombinant Chinese hamster ovary cells." Thesis, University of Kent, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262373.
Full text
7
Gounari, F. "DNA methylation and gene-expression in Chinese hamster ovary cells." Thesis, Imperial College London, 1986. http://hdl.handle.net/10044/1/38022.
Full text
8
Renner, Wolfgang Andreas. "Genetic engineering of the cell cycle regulation of Chinese hamster ovary cells /." [S.l.] : [s.n.], 1995. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=11056.
Full text
9
Spanos, Jonathon L. "Characterisation of IGFBP-5 protease activity in Chinese hamster ovary cells /." Title page and contents only, 2002. http://web4.library.adelaide.edu.au/theses/09SB/09sbr729.pdf.
Full text
10
GLASS, JAMES RUSSELL. "POLYAMINE-MEDIATED DEGRADATION OF ORNITHINE DECARBOXYLASE IN CHINESE HAMSTER OVARY CELLS." Diss., The University of Arizona, 1987. http://hdl.handle.net/10150/184002.
Full textAbstract:
The objective of this research was to identify specific mechanisms involved in the regulation of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway. Immunochemical techniques were used to study post-translational modifications of the ODC protein in relation to activity alterations. Initial experimentation showed that Chinese hamster cells maintained in a defined medium express an ODC protein stable to intracellular degradation. Treatment of these cells with exogenous ornithine or polyamines resulted in a rapid loss of enzyme activity, without detectable changes in the enzyme specific activity. The loss of enzyme activity was a result of accelerated ODC degradation, as determined by immunoprecipitation of pre-labeled protein. In addition, spermidine, but not ornithine, totally inhibited new ODC synthesis. The mechanism of accelerated ODC degradation was investigated and found to occur by an apparent novel mechanism. Degradation of ODC was both ubiquitin-independent and non-lysosomal, and there was also no detectable accumulation of a modified form of ODC protein. In addition, it was found that a component of protein synthesis is required for this process, as inhibitors (cycloheximide, emetine, puromycin) blocked polyamine-accelerated degradation. ODC cDNA was used to synthesize both ODC specific mRNA and protein using in vitro synthesis. These systems may allow the generation of sufficient quantities of material which can be used to recreate in vitro the specific components involved in polyamine inhibition of ODC synthesis and the protease(s) responsible for degradation. The major finding of this work is the direct demonstration that ODC is a stable intracellular protein in the absence of putrescine and spermidine depleted cells (Chapter 2). In addition, that degradation occurs by a novel mechanism, with a requirement for some component of protein synthesis (Chapter 3). Finally, these studies describe the in vitro production of ODC protein and mRNA, which should facilitate further studies of polyamine regulation of ODC degradation and synthesis (Chapter 4).
11
Warner, Richard Gareth. "The expression of #alpha#1,3-galactosyltransferase in Chinese hamster ovary cells." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308706.
Full text
12
Bennett, Claire Michelle. "Kinetic modelling of recombinant IgG2 biosynthesis in Chinese hamster ovary cells." Thesis, University of Sheffield, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548539.
Full text
13
Zhang, Ye. "High cell density perfusion process development for antibody producing Chinese Hamster Ovary cells." Doctoral thesis, KTH, Industriell bioteknologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-207600.
Full textAbstract:
Perfusion operation mode is currently under fast expansion in mammalian cell based manufacturing of biopharmaceuticals, not only for labile drug protein but also for stable proteins such as monoclonal antibodies (mAbs). Perfusion mode can advantageously offer a stable cell environment, long-term production with high productivity and consistent product quality. Intensified high cell density culture (HCDC) is certainly one of the most attractive features of a perfusion process due to the high volumetric productivity in a small footprint that it can provide. Advancements in single-use technology have alleviated the intrinsic complexity of perfusion processes while the maturing in cell retention devices has improved process robustness. The knowledge for perfusion process has been gradually built and the “continuous” concept is getting more and more acceptance in the field. This thesis presents the development of robust perfusion process at very high cell densities in various culture systems. Four HCDC perfusion systems were developed with industrial collaborators with three different mAb producing Chinese Hamster Ovary (CHO) cell lines: 1-2) WAVE Bioreactor™ Cellbag prototype equipped with cell separation by hollow fiber filter utilizing Alternating Tangential Flow (ATF) and Tangential Flow Filtration (TFF) techniques; 3) Fiber matrix based CellTank™ prototype; 4) Glass stirred tank bioreactor equipped with ATF. In all the systems, extremely high viable cell densities above 130 million viable cells per milliliter (MVC/mL) up to 214 MVC/mL were achieved. Steady states were maintained and studied at 20-30 MVC/mL and 100-130 MVC/mL for process development. Perfusion rate selection based on cell specific perfusion rate (CSPR) was systematically investigated and exometabolome study was performed to explore the metabolic footprint of HCDC perfusion process.QC 20170523
14
Haldankar, Raj. "Production of human growth hormone antagonist (hGHG120R) in Chinese hamster ovary cells." Ohio : Ohio University, 1997. http://www.ohiolink.edu/etd/view.cgi?ohiou1174617171.
Full text
15
Wei, Cuihong. "Transcriptional regulation of the asparagine synthetase gene in Chinese hamster ovary cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0007/NQ41335.pdf.
Full text
16
Green, Nicola Helen. "The glycosylation of recombinant human interferon-gamma in Chinese hamster ovary cells." Thesis, University of Kent, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245661.
Full text
17
Geoghegan, Darren. "Characterisation of amino acid transport processes in Chinese Hamster Ovary (CHO) cells." Thesis, University of Sheffield, 2015. http://etheses.whiterose.ac.uk/12970/.
Full textAbstract:
Amino acid transport is controlled by a system of transporter proteins whose function remains poorly defined in CHO cells. In this thesis, the relationship between amino acid transporter activity and culture performance parameters was investigated and used as a basis to explore new opportunities for improving cell line productivity. Transcriptomic analysis of transporter expression was first performed in two non-producing CHO cell lines and one antibody-producing CHO cell line during fed-batch cultures. Seven transporters were highly expressed across the three cell lines (ASCT1, CAT-1, GLAST, LAT1, SNAT2, xCT and y+LAT2). For all cell lines, the cystine-glutamate xCT transporter was significantly upregulated at stationary phase and formed part of a larger adaptive response to support the cellular availability of glutathione. The antibodyproducing cell line also upregulated ASCT1 and LAT1 transporter expression during stationary phase, which allowed the cells to maintain a high consumption rate of amino acids abundant in the antibody. Cells were next treated with inhibitors that block amino acid transport through individual or groups of transporters. Inhibition results confirmed that xCT transport activity is a key determinant of culture viability in all cell lines, and along with LAT1, also supports specific productivity during late-stage culture in the antibody-producing cell line. A directed evolution strategy was subsequently developed to increase xCT transport capability in the host. Evolved host cells demonstrated an increased capacity for GSH synthesis, increased resistance to a ROS insult, and a heritable improvement in cell growth but were not able to outperform the unevolved host in the transient production of an IgG. Finally, a mechanistic model to describe essential amino acid transport processes in CHO cells was constructed from transcriptomic and inhibitor data. This model can be used to direct future optimisation of amino acid concentrations in culture media to maximise cell growth and antibody production.
18
Mozley, O. L. "A mechanistic dissection of polyethylenimine mediated transfection of Chinese hamster ovary cells." Thesis, University of Sheffield, 2013. http://etheses.whiterose.ac.uk/5593/.
Full textAbstract:
Biopharmaceutical production through transient gene expression (TGE) is used within industry for the rapid supply of product for early stage testing. A key requirement of the process is the large scale transfection of mammalian cells, for which the cationic polymer, polyethylenimine (PEI), is widely used. In this thesis, the mechanism of PEI mediated transfection of CHO-S cells is explored at the cell surface, a fundamental barrier to successful transgene delivery. By approaching the question from first principles, exploring the kinetics of transfection at the cell surface, bio-physical and bio-molecular interactions governing polyplex binding to the cell surface, three key findings were made. Firstly, polyplex uptake was biphasic. Initial, rapid endocytosis of polyplex and heparan sulphate proteoglycans (HSPG) was followed by a slower phase of polyplex uptake, on depletion of cell surface HSPGs. Enzymatic depletion of cell surface HSPGs was found to reduce TGE by 25%, whereas sequestration of cholesterol using methyl-β-cyclodextrin abrogated TGE. Taken together, the data indicate that HSPGs mediate maximal TGE (via an early, rapid phase of endocytosis) but that the predominant mechanism of polyplex uptake is through the clustering of lipid rafts, occurring at depleted cell surface HSPG levels. Secondly, the role of both electrostatic and hydrophobic interactions in polyplex binding to the cell surface was investigated. These experiments revealed that at statistically optimized conditions for TGE (with respect to PEI:DNA ratio) the net charge of the polyplex in chemically defined medium was approximately neutral. Under these conditions polyplexes bound to the cell surface, predominantly, via a hydrophobic interaction, independent of cell surface HSPGs. Accordingly polyplex binding to the cell surface was disrupted by both non-ionic surfactant and depletion of plasma membrane cholesterol by methyl-β-cyclodextrin. An increase in polyplex zeta potential at elevated polyplex PEI:DNA ratio increased polyplex binding to the cell surface, but was accompanied by increased cytotoxicity with elevated PEI internalization. A decrease in polyplex zeta potential using ferric (III) citrate resulted in decreased polyplex binding to the cell surface. Both alterations in polyplex charge reduced TGE. Taken together, these data indicate that hydrophobic binding of polyplexes to cell surface lipid rafts (bearing passenger HSPGs) is the primary molecular interaction that promotes subsequent lipid raft clustering and polyplex micro/macropinocytosis to facilitate maximal TGE. Lastly, in order to engineer increased binding and endocytosis of recombinant DNA, alkylated PEIs varying in alkyl chain length and degree of substitution were chemically synthesized in order to increase polyplex hydrophobicity. Compared to unmodified PEI in TGE processes, optimized by Design of Experiments Response Surface Modelling, propyl-PEI was found to mediate more efficient TGE at similar reporter gene titre via a reduction in plasmid DNA load. Propyl-PEI formed polyplexes were found to mediate enhanced polyplex uptake relative to polyplexes formed of unmodified PEI.
19
Kyriakopoulos, Sarantos. "Amino acid metabolism in Chinese hamster ovary cell culture." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/25143.
Full textAbstract:
The present thesis focuses on amino acids (a.a.) and their metabolism by Chinese hamster ovary cells, the workhorse of the multibillion dollar biopharmaceutical industry. The aim of the research was to explore a.a. transport and metabolism and define optimal operating conditions during fed-batch culture, which is the most common process mode used industrially. A fast and reliable way to calculate a.a. concentration ranges in media and feeds is of vital importance, as a.a. are the monomers of proteins, which account for 70% of dry cell weight. The desired recombinant product of bioprocesses is typically also a protein. The transport of a.a. into the cells was studied at the mRNA level of a.a. transporters for the first time in a bioprocessing context. The presented results demonstrate that a.a. transport is not the limiting step for recombinant protein formation. Also, the study allowed for a staged feeding strategy to be designed, where a.a. were not fed altogether. Following linear projection of an integral of viable cell concentration target and using the specific a.a. consumption rates during batch culture, six feeds were formulated containing a.a. and glucose. Three designs were based on the results of the a.a. transport study; however, they underperformed in comparison to the other feeds. In the latter, all nutrients were fed at the same time, resulting in cell culture performance comparable to that obtained with a commercial feed that was tested in parallel. This renders the presented method the first to define a traceable quantitative way to calculate amount of nutrients in the feeds. Flux balance analysis, a powerful technique that allows for investigation of intracellular dynamics, was used to analyse the metabolic data. An enhanced intracellular network was created by coupling two pre-existing in the literature that also for the first time included the glycosylation of the host proteins in the biomass equation. Finally, a novel methodology was developed and coded in R to calculate specific rates of consumption/production of various metabolites in cell culture. The methodology couples mass balances for fed-batch culture operation with constructed vectors of the sampling and feeding schemes. This can be further developed to a bioprocess relevant software platform for analysing cell culture data.
20
Bailey, Laura. "Investigating the influence of long-term culture and feed additions on recombinant antibody production in Chinese hamster ovary cells." Thesis, University of Manchester, 2011. https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-influence-of-longterm-culture-and-feed-additions-on-recombinant-antibody-production-in-chinese-hamster-ovary-cells(1ccfdb8f-c0a6-49c8-a7a7-5e79b84e2862).html.
Full textAbstract:
Chinese hamster ovary (CHO) cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies (MAbs), due to their ability to perform correct post-translational modifications. A major issue for use of CHO cells lines for the production of recombinant proteins is the selection of cell lines that do not retain stable protein expression during long-term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval. Protein production is complex and is influenced by cell growth, transcription, translation, protein folding and post-translational processing and secretory events, which may interact to determine stability of expression during prolonged culture. This thesis aims to identify features associated with stability/instability of recombinant protein expression and methods to improve protein production, with the addition of chemically defined (CD) feed and chemicals. Two exemplar CHO cell lines, which secrete the same recombinant antibody were characterised in response to LTC, feed and DMSO addition. Both cell lines (3.90 and 51.69) exhibited unstable protein production over LTC, with a loss in final antibody titres and specific productivity (Qp). The instability observed within the exemplar cell lines was not due to decreased recombinant gene copy numbers or mRNA expression but was associated with lower viable cell densities, increased ER stress (GADD153 and spliced XBP-1 [XBP-1(s)]) and enhanced rates of lactate utilisation (observed during the decline phase of batch culture). Improvement of recombinant protein expression in response to feed or DMSO addition was associated with lower expression of ER stress markers (ATF4, XBP-1(s) and GADD153 at mRNA level and GADD153 at protein level) and alterations to the metabolic activity of the cultures (prevention of alanine and lactate re-utilisation, and greater glucose utilisation between the stationary and decline phase of batch culture).Although feed or DMSO addition improved recombinant protein production, these additions did not reverse the appearance or progression of instability for cells after LTC. ER stress expression was not abolished as a consequence of feed or DMSO addition. Expression of stress markers at earlier time points may be the factor that limits antibody production and secretion. The consequences of the presence of feed and DMSO addition on ER stress markers and antibody production serves to highlight approaches that may be utilised for engineering more productive or stable protein production phenotypes in parental cell lines.
21
De, Villiers Ann-Marie. "Production and glycosylation of a recombinant protein from Chinese hamster ovary (CHO) cells." Thesis, Stellenbosch : Stellenbosch University, 2012. http://hdl.handle.net/10019.1/71663.
Full textAbstract:
Thesis (MScEng)--Stellenbosch University, 2012.ENGLISH ABSTRACT: Recombinant glycoproteins are important biopharmaceuticals, providing solutions for numerous previously untreatable illnesses, in everything from cancer to infertility. Most recombinant biopharmaceuticals are produced in mammalian cells due to their ability to provide the correct post-translational processing for use in humans. The post-translation processing influences many of the protein’s properties including pharmacokinetics, bioactivity, secretion, half-life, solubility, recognition and antigenicity. The aim of this thesis is to further study the upstream production of a glycosylated recombinant protein produced by Chinese hamster ovary (CHO) cells on production scale within the confines of an existing process. The process in question uses adherent CHO cells to produce a glycosylated recombinant hormone. As with most recombinant protein production processes, this process has two sections to the upstream production: a seed train to grow enough cells to inoculate production, and a production section, which focuses on the production of a recombinant protein. The seed train is predominantly conducted in roller bottles, while the production section takes place in perfusion bioreactors, where the cells are attached to microcarriers, with spin-filters for cell retention. The whole process uses medium with serum. There are two process challenges regarding an existing recombinant-protein production process: 1. The gradual increase, over the past several campaigns, of the final population doubling level of the cells (which must remain within certain specified limits) at the end of the seed train. 2. The low glycosylation levels of the product seen in certain campaigns, which meant that a certain number of final product batches were below the specified acceptable glycosylation limits. Following a literature survey several controlled process variables were chosen for investigation and hypotheses made on their effect on the seed train or glycosylation. To investigate their effect on the PDL and cell growth in the seed train: - Medium volume: decreasing the medium volume will yield a lower PDL due to slower cell growth caused by lower glucose availability. - Seeding density: if cells obtain confluence by the time they are harvested, decreasing the seeding density will yield a higher PDL. - Cultivation temperature: decreasing the temperature ought to decrease the growth rate. - Medium feed temperature: there will be no significant difference to the cell culture when pre-heated or cold medium is used. Aeration: using vent caps will increase the oxygen content of the medium in the roller bottles and the cell growth, yielding a higher PDL. To investigate their effect on glycosylation during production: - pH: better glycosylation will be seen at pH 6.9, than at pH 6.7. - Perfusion rate: a higher perfusion rate will lead to better glycosylation due to increased glucose and glutamine concentrations. In the seed train, the only factor that significantly influenced the final PDL was the seeding density. Cell growth was inhibited once cells reached confluence, so lowering the seeding density lead to a higher PDL. It is recommended to use a high seeding density to ensure a lower PDL. Historic data indicated that the seeding density was not the cause of the apparent increase of the final PDL, as all previous campaigns had been seeded with a high seeding density. What then became apparent was that the final PDL remained relatively constant during a campaign and that the increase in final PDL occurred between campaigns. It appears that the apparent increase in the final PDL is due to differences in cell counting between operators as each new campaign was managed by different operators. It is recommended that a mechanical cell counter be used to verify cells counts and to maintain a standard between campaigns. In the bioreactors, varying the pH proved to have no significant effect on the glycosylation levels. However, both the initial perfusion rate and the specific perfusion rate proved to be important from both historical data and the data generated during these experiments. Lower levels of the initial perfusion rate lead to better glycosylation and it is recommended that an initial perfusion rate of 1.0 volumes/day be used. The relationship between the specific perfusion rate and the glycosylation appears to be non-linear and requires further study, for now it is recommended that the specific perfusion rate be kept below 0.3 volumes/day/109 cells. Probable reasons for the unsatisfactory glycosylation seen in certain runs could also be proposed from these two factors: • RP33-133 : Very high specific perfusion rate • RP32-135 : High initial perfusion rate and very high specific perfusion rate • RP32-138 : High initial perfusion rate • RP33-139 : High initial perfusion rate Further research is recommended into the effect of the specific perfusion rate as well as the specific glucose consumption rate and the specific glutamine concentration on the glycosylation.
AFRIKAANSE OPSOMMING: Rekombinante glikoproteïene is baie belangrike biofarmaseutiese produkte wat oplossings bied vir talle voorheen ongeneeslike siektes in alles van kanker tot onvrugbaarheid. Meeste rekombinante farmaseutiese produkte word gemaak deur diere-selle as gevolg van hulle bevoegtheid om die korrekte na-translasie stappe te volg sodat die produkte in mense gebruik kan word. Die na-translasie stappe beïnvloed baie van die proteïene se karaktertreke insluitende die farmakokinetika, bioaktiwiteit, uitskeiding, half-leeftyd, oplosbaarheid, herkenbaarheid and antigeniciteit. Die doel van hierdie tesis is om die stroomop produksie van ‘n rekombinante glikoproteïene vervaardig deur Chinese hamster ovariale (CHO) selle verder te bestudeer binne die grense van ‘n bestaande proses op grootskaalse vlak. Die huidige proses gebruik CHO selle om ‘n rekombinante glikohormoon te produseer. Soos meeste prosesse wat rekombinante proteïene produseer bestaan die stroomop gedeelte van die proses uit twee dele: ‘n saad trein wat genoeg selle maak vir produksie en ‘n produksie gedeelte wat fokus op die vervaardiging van die glikoproteïen. Die saad trein bestaan hoofsaaklik uit roller bottels terwyl produksie plaasvind in perfusie bioreaktors waar die selle op “microcarriers” groei, met spin-filters om die selle binne die bioreaktors te hou; die hele proses gebruik medium met serum. Daar is twee probleme in die stroomop gedeelte van die bestaande proses: 1. Die geleidelike toename oor die afgelope paar jaar van die finale verdubbelingsvlak van die selle aan die einde van die saad trein 2. Die lae glukosilering van die eindproduk wat veroorsaak dat sekere lotnommers buite spesifikasie is Na ‘n literatuur studie, was seker beheerde proses parameters gekies om verder te bestudeer en hipotesisse gemaak oor hulle effek op die saad trein of die vlak van glukosilering. Die volgende faktore is bestudeer vir hulle effek op die finale verdubbelingsvlak van die selle in die saad trein: - Medium volume: ‘n laer medium volume sal lei tot a laer verdubbelingsvlak van die selle as gevolg van stadige groei - Konsentrasie van selle vir inokulasie: as die selle konfluent is teen die tyd wat hulle versamel word sal ‘n laer konsentrasie selle lei tot ’n hoër verdubellingsvlak. - Temperatuur: laer temperatuur behoort te lei tot ‘n stadiger groei koers van die selle - Medium voer-temperatuur: die voer-temperatuur van die medium sal geen beduidende verskil maak - Belugting: die gebruik van “vent-caps” sal die suurstof inhoud van die roller bottels verhoog Die volgende faktore is bestudeer vir hulle effek op die glukosilering tydens produksie: - pH: beter glukosilering word verwag by by pH 6.9 dan by pH 6.7 - Perfusie koers: ‘n hoër perfusie koers sal lei tot beter glukosilering as gevolg van hoër glukose en glutamien konsentrasies Die konsentrasie van die selle wat gebruik word vir inokulasie blyk die enigste faktor te wees wat die finale verdubbelingsvlak van die selle en die groei van die selle in die saad trein beïnvloed het. Die groei van die selle was beprek wanneer die selle konfluent geraak het en dus het ‘n laër sel konsentrasie by inokulasie gelei tot ‘n hoër sel verdubbelingsvlak. Dit word aanbeveel dat ‘n hoë sel konsentrasie by inokulasie gebruik word. Die geleidelike toename van die finale verdubbelingsvlak van die selle in die saad trein is waarskynlik as gevolg van die variasie in sel tellings tussen verskillende operateurs eerder as as gevolg van die beheerde proses parameters. Dit word aanbeveel dat ‘n meganiese sel-teller gebruik word om die verskil in sel tellings tussen operateurs te kontroleer en om ‘n standaard te handhaaf tussen produksie lotte. In die bioreaktors, het die pH geen beduidende invloed gehad op die glukosilering maar uit historiese data en die huidige data van hierdie eksperimente blyk albei die begin perfusie koers en die spesifieke perfusie koers ‘n belangrike invloed te hê op die glukosilering. Laër vlakke van die begin perfusie koers lei tot beter glikosilsie en dit word aanbeveel dat elke produksielot ‘n begin perfusie koers het van 1.0 volume/dag. Die verhouding tussen die glukosilering en die spesifieke perfusie koers blyk om nie-liniêr te wees nie. Nog navorsing hieroor word aanbeveel, maar vir nou word dit aanbeveel dat die spesifieke perfusie koers onder 0.3 volumes/dag/109 selle gehou word. Hierde twee faktore blyk die oorsaak te wees vir die lae glukosilering wat in sekere produksielopies gevind was: • RP33-133 : baie hoë spesifieke perfusie koers • RP32-135 : hoë begin perfusie koers en baie hoe spesifieke perfusie koers • RP32-138 : hoë begin perfusie koers • RP33-139 : hoë begin perfusie koers Dit word aanbeveel dat verdere navorsing gedoen word op die effek van die spesifieke perfusie koers asook die spesifieke koers van glukose verbruik en die spesifieke glutamien konsentrasie op die glukosilering van die produk.
22
Adams, K. "Studies on the resistance to antimicrotubular agents in cultured Chinese hamster ovary cells." Thesis, University of York, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377298.
Full text
23
Fenton, James A. L. "The relationship between protein kinases and multidrug resistance in Chinese hamster ovary cells." Thesis, University of York, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336842.
Full text
24
Arnall, Claire Lucy. "High-throughput platform development for multigene engineering of Chinese Hamster Ovary (CHO) cells." Thesis, University of Sheffield, 2018. http://etheses.whiterose.ac.uk/20994/.
Full text
25
Nyberg, Gregg B. (Gregg Bartell) 1970. "Glycosylation site occupancy heterogeneity in Chinese hamster ovary cell culture." Thesis, Massachusetts Institute of Technology, 1998. http://hdl.handle.net/1721.1/28190.
Full text
26
Yang, Lin. "The signaling pathway of oxysterol induced apoptosis in Chinese hamster ovary (CHO)-K1 cells." [Johnson City, Tenn. : East Tennessee State University], 2002. http://etd-submit.etsu.edu/etd/theses/available/etd-0510102-161626/unrestricted/YangL062602.pdf.
Full text
27
Johnson, Nicholla Rachael. "Phosphoinositide and Ca2+ signalling in Chinese hamster ovary cells expressing recombinant muscarinic M3 receptors." Thesis, University of Leicester, 1996. http://hdl.handle.net/2381/33630.
Full textAbstract:
It is well established that M3 muscarinic receptors couple, via the Gq/11 family of G-proteins, to the activation of phosphoinositidase C (PIC) leading to Ins(l,4,5)P3 formation and intracellular Ca2+ mobilisation (Caulfield, 1993). The ability of many muscarinic agonists to initiate a response via the phosphoinositide pathway is known to be tissue dependent. Using recombinant human muscarinic M3 receptors, expressed as a homogeneous population in a host CHO-Kl cell line, the significance of receptor-G-protein stoichiometry, in agonist mediated inositol phosphate and Ca2+ responses, was investigated. Agonist-stimulated PIC and Ca2+ responses in cell lines expressing different densities of the M3 receptor, or cells treated with an alkylating agent, were compared in an attempt to determine the relationship between M3 muscarinic occupancy by full and partial agonists and the efficiency of coupling to the effector PIC. The transfected cell lines appeared to have a classical 'reserve' for PIC activation, which was dependent on receptor density and partly dependent on extracellular Ca2+ concentration. This reserve appeared to be greater when PIC activity was assessed by total [3H]inositol phosphate accumulation rather than Ins(l,4,5)P3 mass accumulation, indicating that the method of assessment must be taken into account when determining the characteristics of phosphoinositide signalling. Agonists appeared to be more efficient at mobilising intracellular Ca2+ than activating PIC, as dose response curves for agonist-stimulated Ca2+ mobilisation lay to the left of those for PIC activation. The ability of agonists to mobilise intracellular Ca2+ also appeared to be dependent on receptor density and extracellular Ca2+ concentration. However, there did not appear to be a receptor reserve for this response. In conclusion, the results obtained for the PIC response appear to follow the predictions of classical receptor theory and Ca2+ mobilisation, as a response downstream of PIC activation, is more sensitive to agonists, as would be expected following an amplification of the signal. However, following amplification, an increase in receptor reserve would be expected. Clearly this is not the case and the agonist induced increases in intracellular Ca2+ in this study cannot be adequately described by classical receptor theory.
28
Brewer, F. "Resistance to the vinca alkaloids in Chinese hamster ovary cell lines." Thesis, University of York, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380511.
Full text
29
Murray, Richard C. "The reduced folate carrier gene in Chinese hamster ovary cells, gene characterization and mutant analysis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq28508.pdf.
Full text
30
Croxford, Alexandra Sarah. "Optimisation of recombinant protein production in Chinese hamster ovary cells using ubiquitous chromatin opening elements." Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.493616.
Full textAbstract:
The requirement for complex, therapeutic proteins that possess the correct post-translational modifications has resulted in mammalian cells, in particular Chinese hamster ovary (CHO) cells, being widely used in die biopharmaceutical industry, In the creation of mammalian cell lines plasmid DNA carrying the gene of interest integrates randomly into die host cell genome. Integration into different chromatin domains results in variable levels of gene expression between cell lines due to gene silencing mechanisms. In addition, cell lines often show unstable protein production during long-term culture. Variable and unstable recombinant protein expression means that a large number of clones need to be screened in order Therefore there is a necessity to overcome these gene silencing mechanisms, with the intention to accelerate process development of recombinant protein production in mammalian systems.
31
Glynn, Anne-Marie. "Cryo-electron tomography of frozen-hydrated sections of supramolecular complexes in Chinese Hamster Ovary Cells." Thesis, University of Dundee, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505634.
Full text
32
Underhill, Michele F. "Engineering mRNA translation initiation in Chinese hamster ovary cells for enhanced production of recombinant proteins." Thesis, University of Kent, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322824.
Full text
33
Patel, Tulshi. "Manipulation and exploitation of microRNAs for enhanced recombinant protein production in Chinese hamster ovary cells." Thesis, University of Kent, 2017. https://kar.kent.ac.uk/66224/.
Full textAbstract:
MicroRNAs (miRs) are a class of non-coding RNA that function to regulate global mammalian gene expression by mediating the translational repression of mRNAs harbouring a complementary target region sequence within their 3'UTR. The RNA-RNA binding event between the miR seed region and mRNA target region disrupts mRNA translation and hence repression of protein synthesis from multiple mRNA targets encompassing a variety of cellular processes and pathways. In addition to this multiplicity, miRs also exhibit a high level of promiscuity as a single miR may silence many mRNA targets and a single mRNA transcript may be under the regulation of multiple miRs. miR activity may therefore be manipulated to favour/inhibit cellular processes/pathways of interest to yield desirable cell phenotypes such as enhanced secretion, metabolism or growth. The engineering of miR activity has been applied to enhancing the production of recombinant proteins in the industrially-relevant Chinese hamster ovary (CHO) cell line. In the industrial setting, the utilization of CHO cells has become the dominant mammalian system for manufacturing biotherapeutic recombinant proteins due to their aptitude for accurate protein folding, assembly and performing 'human like' post-translational modifications. A historical Lonza microarray conducted on a range of GS-CHOK1SV IgG-producing cell lines identified changes in miR abundance throughout culture that correlated with growth or recombinant IgG productivity. From this screen, three miRs in particular (miR-15b, -16-1 and -34c) were selected for further study with regard to the impact of targeted miR knockdown and engineered pri-miR over-expression in recombinant and host CHO cell lines and transfectants with respect to establishing whether changes in the amounts of these miRs impacted upon CHO cell growth and productivity. The studies undertaken here have shown that the engineered over-expression of pri-miRs can improve the longevity (e.g. when over-expressing pri-miR-16-1-34c and -15b-34c-16-1) and maximum viable cell concentration (e.g. when over-expressing pri-miR-15b, -16-1 and -16-1-34c) of a CHOK1SV-GSKO host cell line whilst the expression of miR-sponges for targeted miR knockdown can enhance the cell specific productivity (e.g. when expressing miR-sponge-S6) in a variety of GS-CHOK1SV IgG-producing cell lines. In particular, miR-sponges derived from the 3'UTR of the SRPRα mRNA, which should be targeted by miR-34c, reduced miR-mediated repression of SRPRα expression and hence an increase in SRPRα mRNA and protein expression was observed. Specifically, the application of a SRPRα-derived miR-sponge construct constituting 6 miR-binding site motifs was shown to be both functional in relieving endogenous SRPRα from miR-mediated translational repression as well as potentially enabling an increase in cell specific productivity in selected recombinant CHO cell lines, suggesting that secretory capacity was limited by the availability of SRPRα. In conclusion, the studies presented here have demonstrated that the exploitation of miR activity can be an effective tool for CHO cell engineering for the tuning of recombinant protein production without placing any additional translational burdens on the cell.
34
Mekonnen, Tsehay Eshete. "Induction of heat shock protein 70 in Chinese hamster ovary cells during chlamydia trachomatis infection." CSUSB ScholarWorks, 1994. https://scholarworks.lib.csusb.edu/etd-project/2969.
Full text
35
Sullivan, Peter C. "Studies on the internalization and intracellular transport of horseradish peroxidase in Chinese hamster ovary cells." Diss., Virginia Polytechnic Institute and State University, 1985. http://hdl.handle.net/10919/49910.
Full textAbstract:
Soluble horseradish peroxidase (HRP) is internalized by Chinese hamster ovary cells, a cell line of fibroblastic origin (Adams et al., 1982). We have confirmed this result by showing no inhibition of uptake in the presence of divalent cation chelators (EGTA Mg or EDTA), excess (19 mg/ml) yeast mannan (an inhibitor of uptake through a mannose/N-acetylglucosamine receptor) or using periodate treated HRP. Periodate treatment destroys the ring structure of sugars on HRP which have hydroxyl groups on adjacent ring carbons, eliminating sugar mediated uptake of HRP. Once internalized, HRP is found in endocytic vesicles which by HRP-cytochemical staining, show deposits which rim the luminal face of vesicle membrane. Once HRP is in lysosomes, cytochemical deposits are luminal. To test if HRP is actually associated with vesicle membrane, a hypotonic lysis assay was used. Postnuclear supernatants (PNS) from cells pulse labeled with HRP were lysed and the percent of HRP sedimenting with a high speed membrane fraction was used as a measure of membrane association. After a pulse, >60% of the total HRP internalized was pelletable. Hypotonic lysis of a PNS at different pH and temperature showed no significant difference in "pelletability" from 4℃ to 37℃ at neutral pH and only a slight decrease in "pelletability" with increased temperature (4℃ to 37℃) at pH M.6. Binding of HRP in a membrane preparation was pH and temperature stable. Uptake of native HRP in the presence of yeast mannan (19 mg/ml) or using periodate treated HRP also had little effect on "pelletability", suggesting the absence of sugar specific binding in endocytic vesicles. Using the hypotonic lysis assay of a PNS after different chase times, HRP dissociation from membrane was observed over a 30 minute chase period. Internalized HRP in the presence of yeast mannan (19 mg/ml), intravesicular pH elevators HEPES (40 mM) or monensin (10 μM), or substances which should deplete cellular ATP NaF/KCN (2 mM /1 mM), showed no inhibition of dissociation kinetics. A chase at 17℃ inhibited dissociation of HRP over the entire 30 minute period. This HRP binding site(s) appears unique to endocytic vesicles.
A minimum of four steps in transport have been identified based on their sensitivity to inhibitors. HRP transport, identified by Percoll density gradient fractionation, was inhibited at 17°C and was sensitive to pH elevators (NH₄Cl, monensin, HEPES) and ATP depletion (NaF/KCN). Inhibition of transport appeared to be independent of HRP dissociation except at early temperature sensitive step(s). These results suggest that transport inhibition may be due to an effect on a) inhibition of membrane dissociation (early step(s)) and alteration of membrane fluidity (later steps) by reduced temperature and b) transmembrane events by pH elevators and ATP depletion.Ph. D.
incomplete_metadata
36
Pybus, Leon P. "Engineering the expression of "difficult-to-express" recombinant monoclonal antibodies in Chinese hamster ovary cells." Thesis, University of Sheffield, 2014. http://etheses.whiterose.ac.uk/6638/.
Full text
37
Walther, Christa G. "Adaptation to suspension growth : analysing the surface of suspension growth adapted Chinese hamster ovary cells." Thesis, University of Sheffield, 2013. http://etheses.whiterose.ac.uk/4193/.
Full textAbstract:
Many bio-pharmaceutical production processes are based upon the use of mammalian cell lines, such as Chinese hamster ovary (CHO) cells, capable of proliferation as single cells in suspension in a synthetic environment. Routine use of CHO cells as production vehicles requires a lengthy “adaptation” process from the wild-type adherent clone to clones capable of proliferation in a suspension environment depleted of exogenous growth factors and cell-matrix contacts. Different approaches have been applied in this study to gain a better understanding of the changes on the cell surface occurring as a response to changes in their environment, comparing four cell lines (CCL61, AML, S cells and CHO-S) adapted to suspension or adherence growth condition. Biochemistry and mass spectrometry methods showed differences in surface protein composition for the cell lines. The comparison of the expression of cell-to-cell adhesion molecules revealed a highly variable bimodal distribution on S cells which was not seen on CCL61. Analysis of the expression level of integrins, the main interaction partner of serum components, indicated that integrin expression is not generally down-regulated on suspension-adapted CHO cells. The integrin conformation on the cell surface, analysed by confocal microscopy, revealed a specific conformation, especially with regard to integrin beta 1, characterised by an even, net-like distribution of integrin clusters over the surface of the cells. This specific integrin conformation, which has only been found on suspension-adapted cells, was underlined by a sub-cortical sheet of actin, forming a ball-like structure directly under the cell membrane; this actin conformation required re-organisation of the actin cytoskeleton from a typical fibrillar morphology in adherent cells. The actin content was higher in suspension cell lines compared to adherent cells, but actin up-regulation was also found in non-suspension adapted cells after they had been transferred into suspension. Sphere-like integrin beta 1 clustering on CCL61 grown in suspension could be induced by treatment with cytochalasin D, followed by suspension culture without the drug, however, despite the change in integrin beta 1 conformation these cells could not grow in suspension. The data suggests that adaptation to suspension growth requires conservation of integrins, presumably with respect to their role as structural elements anchoring the plasma membrane to the sub-cortical actin sheath, but it also requires additional changes in the interplay between integrin beta 1 and actin, for example, changes in the regulation of the associated proteins for successful suspension adaptation of CHO cells.
38
Lim, Yiping. "Elucidating & targeting apoptotic genes in Chinese hamster ovary (CHO) cell culture." Thesis, Imperial College London, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.537560.
Full text
39
Brown, Adam. "Tools for next-generation transcriptional control in Chinese hamster ovary cell factories." Thesis, University of Sheffield, 2014. http://etheses.whiterose.ac.uk/7335/.
Full textAbstract:
Recombinant gene transcription in Chinese hamster ovary (CHO) cells, the dominant cell factory utilised for biopharmaceutical production, is still routinely regulated with a limited set of functionally ill-defined and uncontrollable genetic elements. This study presents novel transcription control technologies that facilitate development of next-generation biopharmaceutical manufacturing systems. Firstly, synthetic promoters designed specifically to harness the pre-existing transcriptional activation machinery of CHO cell factories have been constructed. Transcription factor regulatory element (TFRE) function was screened in CHO cells and active elements were utilised to create synthetic promoter libraries exhibiting 140 discrete activites, operating over two orders of magnitude, where the most active promoters significantly exceeded that of the human cytomegalovirus immediate early 1 (hCMV-IE1) promoter. Through precise control of recombinant gene expression in CHO host cells over a broad dynamic range this technology could be utilised to both maximise transcription of easy-to-express proteins and provide optimised protein-specific transcription levels (synchronised with polypeptide-specific folding kinetics) of difficult-to-express proteins. Further, it will enable construction of bespoke, synthetic cell factories that require the expression of several genes to be stoichiometrically balanced. Secondly, a novel method of transcription factor (TF) decoy (synthetic oligodeoxynucleotides that specifically sequester cognate TFs) formation has been developed, where blocks containing discrete TF binding sites are combined into circular molecules. Unlike currently available methods block-decoys allow rapid construction of chimeric decoys targeting multiple TFREs. Moreover, they enable fine tuning of binding site copy ratios within chimeras, facilitating sophisticated control of the cellular transcriptional landscape. It was demonstrated that a bespoke block-decoy chimera was able to inhibit expression from multiple target elements simultaneously in CHO cells. Block-decoys can be utilised to investigate any multi-TF mediated cell function or phenotype and represent a valuable new tool for characterising and controlling CHO cell transcription. Finally, the mechanistic functionality of the promoter most commonly utilised to drive transgene expression in CHO cells, hCMV-IE1, has been analysed. It was found that hCMV-IE1 promoter activity in CHO cells is predominantly mediated via just two TFREs (CRE and NFkB), where physical prevention of TF-TFRE interactions at these sites, either by intracellular TF sequestration or TFRE deletion, reduced activity by >75%. This mechanistic understanding of hCMV-IE1s functional regulation in CHO cells facilitates strategies to predictably control or improve its activity by engineering the promoter's TFRE composition or the cell factory's TF abundances. This will likely be most useful for optimising transient gene expression systems where hCMV-IE1 is the current promoter of choice. Cumulatively, the tools developed in this thesis enable sophisticated, next-generation transcriptional control in CHO cell factories.
40
Skepu, Amanda. "Identification and characterisation of a novel gene, DWNN, isolated from promoter-trapped Chinese hamster ovary cells." Thesis, University of the Western Cape, 2005. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_7576_1249626665.
Full textAbstract:
The process of cytotoxic T lymphocyte (CTL) killing involves the recognition and destruction of foreign antigens by cytotoxic T cells and is of crucial importance to the defence of the organism against viral infections. Defects in this process can lead to various autoimmune diseases and cancer. The aim of this study was to identify more genes involved in the cell death pathway and to link CTL killing, apoptosis and cancer.
41
Bordon, Harriet. "Wirkung von Inositolphosphatkinase-Inhibitoren auf den Inositolphosphatstoffwechsel und das Wachstum von chinese hamster ovary cells (CHO)." [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=966309243.
Full text
42
Sundaram, Hardy. "Characterisation of recombinant human serotonin 5-HTâ†1â†A receptors expressed in Chinese hamster ovary cells." Thesis, University of Kent, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262508.
Full text
43
Kemp, L. M. "The isolation and characterization of x-ray sensitive mutants of the Chinese hamster ovary cell line." Thesis, University College London (University of London), 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.354357.
Full text
44
Sun, Qian. "Molecular analysis of factors involved in regulation of protein expression by recombinant Chinese hamster ovary (CHO) cells." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505481.
Full textAbstract:
The demand for approved biopharmaceuticals products from animal cell culture have been rapidly increasing since last few decades. Many efforts have been made on process refining in the past, and now, works are focusing on the selection or generation of high producer cell lines. It was therefore very important to understanding the molecular mechanisms underlying the existing high producing cell lines and to identify cellular regulators responsible for enhanced productivity.
45
Strotbek, Michaela [Verfasser], and Monilola A. [Akademischer Betreuer] Olayioye. "MicroRNAs to boost the productivity of Chinese hamster ovary producer cells / Michaela Strotbek. Betreuer: Monilola A. Olayioye." Stuttgart : Universitätsbibliothek der Universität Stuttgart, 2014. http://d-nb.info/1051621666/34.
Full text
46
Godfrey, Charlotte. "Investigation of translational reprogramming during transient and stable expression of monoclonal antibodies in Chinese hamster ovary cells." Thesis, University of Kent, 2017. https://kar.kent.ac.uk/65774/.
Full textAbstract:
Translational reprogramming and mRNA translation e ciency greatly in uence global protein synthesis, cell proliferation and growth; important parameters in de ning recombinant protein expression yields. Polysome pro ling is a widely-used technique to analyse mRNA transla- tion and its e ciency that provides a snapshot of ribosomes loaded on mRNA transcripts at any particular time. A higher number of polysomes present on a given mRNA suggests that the mRNA is being more heavily translated than those mRNAs with few ribosomes. Fur- ther, a large pool of sub-polysomes (40S, 60S and 80S) compared to polysomes in a sample suggests low translational activity. Here, polysome pro ling has been applied to investigate translational reprogramming in multiple recombinant monoclonal antibody (mAb)-producing Chinese hamster ovary (CHO) cell lines, and to determine how reprogramming re ects the ability of such cells to proliferate and make recombinant proteins in stable and transient mAb expression systems, in batch and fed-batch culture mode. The impact of culture temperature on the polysome pro le and hence on reprogramming was also investigated in transient studies. Polysome pro ling revealed reprogramming di ered between recombinant cell lines. Those with the highest global translational e ciency generally had the fastest cell speci c growth rates, although total ribosome capacity did not directly relate to those with the fastest growth rates or mAb productivities. This suggests it is the ability to utilise available machinery that determines protein synthetic capacity. Recombinant cell lines with higher cell speci c produc- tivities generally maintained a higher polysome to monosome (P:M) ratio during stationary phase and had elevated recombinant mRNA copy numbers localised to translationally active heavy polysomes. In transient systems, the P:M ratio was maintained longer at reduced tem- perature cultivation and related to higher mAb yields being obtained. A number of endogenous transcripts were found to be more or less abundant on polysomes at di erent times of culture, indicative of changes in the cellular requirements of the encoded proteins. Such transcripts could be potential cell engineering targets to help tune the needs of the cell to the demands of a culture process or recombinant protein, or alternatively their untranslated regions harnessed to help preferentially load target mRNAs onto ribosomes. When upstream open reading frames (uORFs) or alternative translation start sites were engineered into recombinant transcripts a range of mAb expressions were observed allowing the tuning of mAb expression, including improvement over a standard untranslated region used industrially as a control. The ndings described in this thesis therefore reveal insights into the mechanisms involved in translational regulation and reprogramming in CHO cells during bioprocessing. These can be utilised for further improvement via targeted cell engineering strategies, cell line screen- ing approaches or modi cation of recombinant transcripts for enhanced industrial host and recombinant cell lines.
47
Page, Catherine. "Investigating the consequences of exogenous expression of unfolded protein response components in recombinant Chinese hamster ovary cells." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-consequences-of-exogenous-expression-of-unfolded-protein-response-components-in-recombinant-chinese-hamster-ovary-cells(8f2c791e-b7ce-4b5d-b363-af950a9dab0e).html.
Full textAbstract:
Chinese hamster ovary (CHO) cells are frequently used for the commercial expression of recombinant therapeutic antibodies due to their ability to perform appropriate post-translational modifications and therefore generate an accurate rendition of natural products. As a consequence of their initial derivation by mutagenesis and the divergence into distinct cell lines, clonally-derived cell lines are phenotypically distinct. Molecular understanding of the features that determine the properties of a CHO clone is fundamental to the optimisation of cell environment and, potentially, engineering or selection of CHO clones with the “best” phenotype.The profile of the endoplasmic reticulum (ER) environment (with a specific complement of chaperones, co-chaperones, and sensors) is important for cell growth and maximum recombinant protein secretion. I have addressed how the modulation of two components in the ER, XBP1(s) and ERO1L α, influence CHO cell function. XBP1(s) is generated by a novel mRNA splicing mechanism in response to ER stress and is the key regulator factor for the development of professional secretory cells. ERO1L α plays a critical role in setting the redox state of foldases (such as PDI) and is also known to be induced by ER stress. In this study, CHO S cells were doubly transfected with human XBP1(s) and human ERO1L α constructs to generate a series of CHO cell lines that overexpressed each gene. The engineered cell lines exhibited a series of improvements in terms of desirable phenotypes compared to the non-engineered CHO S cell line. These improvements included up-regulation of chaperone expression, alteration in growth profile and associated glucose consumption and lactate production, increased antibody titres, and improved recovery from an oxidative stress. My interpretation is that engineering cells to over-express XBP1(s) and ERO1L α generated a more favourable phenotype for cells to handle the stresses that result from protein transit in the ER. Whether this is a direct effect of XBP1(s) and ERO1L α or due to a secondary consequence of their over-expression on the ER chaperone complement remains unclear. However, this study identified important combinations of regulatory factors that influence ER function and, consequently, the ability to define improved CHO cell phenotypes for expression of different types of protein products.
48
Nakamura, Tetsuo. "Expression of Glycosylated Human Interferon-beta (IFN-β) in High Levels in Chinese Hamster Ovary (CHO) Cells." Kyoto University, 2000. http://hdl.handle.net/2433/151588.
Full text
49
Allen, Lee-Ann Hill. "Peroxisome biogenesis in Chinese hamster ovary cells." 1990. http://catalog.hathitrust.org/api/volumes/oclc/23535925.html.
Full textAbstract:
Thesis (Ph. D.)--University of Wisconsin--Madison, 1990.Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.
50
Bulseco, Dylan A. "Muscarinic receptor-effector coupling in Chinese hamster ovary cells." Thesis, 1996. http://hdl.handle.net/1957/34134.
Full text