Dissertations / Theses on the topic 'Biopharmaceutical proteins'
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Wei, Tzu-Hsiang Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Transient production of biopharmaceutical proteins." Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences, 2009. http://handle.unsw.edu.au/1959.4/43708.
Full textPradhan, S. "Rational design of enterokinase for the development of enhanced biopharmaceutical proteins." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1378549/.
Full textBatra, Sumit. "Innovative Purification Protocol for Heparin Binding Proteins: Relevance in Biopharmaceutical and Biomedical Applications." TopSCHOLAR®, 2011. http://digitalcommons.wku.edu/theses/1062.
Full textBuyel, Johannes Felix [Verfasser]. "Manufacturing biopharmaceutical proteins by transient expression in Nicotiana tabacum (L.) / Johannes Felix Buyel." Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2013. http://d-nb.info/1043518819/34.
Full textHameed, Rana Majeed. "The application of aqueous two phase systems to the analysis of protein isoforms of importance in clinical biochemistry and biopharmaceutical production." Thesis, Brunel University, 2016. http://bura.brunel.ac.uk/handle/2438/14452.
Full textBuyel, Johannes Felix [Verfasser], and J. [Akademischer Betreuer] Hubbuch. "Overcoming Hurdles in Downstream Processing of Tobacco-derived Biopharmaceutical Proteins / Johannes Felix Buyel ; Betreuer: J. Hubbuch." Karlsruhe : KIT-Bibliothek, 2017. http://d-nb.info/1148551220/34.
Full textDehling, Marco [Verfasser], Johannes [Akademischer Betreuer] Buchner, Matthias [Gutachter] Feige, and Johannes [Gutachter] Buchner. "Conformational analysis of proteins of biopharmaceutical interest / Marco Dehling ; Gutachter: Matthias Feige, Johannes Buchner ; Betreuer: Johannes Buchner." München : Universitätsbibliothek der TU München, 2018. http://d-nb.info/1153545853/34.
Full textDehling, Marco Verfasser], Johannes [Akademischer Betreuer] [Buchner, Matthias [Gutachter] Feige, and Johannes [Gutachter] Buchner. "Conformational analysis of proteins of biopharmaceutical interest / Marco Dehling ; Gutachter: Matthias Feige, Johannes Buchner ; Betreuer: Johannes Buchner." München : Universitätsbibliothek der TU München, 2018. http://nbn-resolving.de/urn:nbn:de:bvb:91-diss-20180216-1404726-1-3.
Full textArfi, Zulfaquar Ahmad Verfasser], Rainer [Akademischer Betreuer] [Fischer, and Stefan [Akademischer Betreuer] Schillberg. "Development of an immunoassay to detect tobacco host cell proteins during biopharmaceutical development / Zulfaquar Ahmad Arfi ; Rainer Fischer, Stefan Schillberg." Aachen : Universitätsbibliothek der RWTH Aachen, 2015. http://d-nb.info/1129176789/34.
Full textArfi, Zulfaquar Ahmad [Verfasser], Rainer [Akademischer Betreuer] Fischer, and Stefan [Akademischer Betreuer] Schillberg. "Development of an immunoassay to detect tobacco host cell proteins during biopharmaceutical development / Zulfaquar Ahmad Arfi ; Rainer Fischer, Stefan Schillberg." Aachen : Universitätsbibliothek der RWTH Aachen, 2015. http://d-nb.info/1129176789/34.
Full textHebditch, Max. "Computational modelling approaches for studying protein-protein and protein-solvent interactions in biopharmaceuticals." Thesis, University of Manchester, 2018. https://www.research.manchester.ac.uk/portal/en/theses/computational-modelling-approaches-for-studying-proteinprotein-and-proteinsolvent-interactions-in-biopharmaceuticals(b965e1ee-0769-476c-970b-6c676468e577).html.
Full textMcGarvey, O. S. "Calorimetric, structural and spectroscopic studies on trehalose as a protein cryoprotectant." Thesis, Queen's University Belfast, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273088.
Full textFung, Ho Ki. "Synthesis and development of manufacturing processes for biopharmaceuticals /." View Abstract or Full-Text, 2003. http://library.ust.hk/cgi/db/thesis.pl?BIEN%202003%20FUNG.
Full textKheddo, Priscilla. "Effect of arginine glutamate on protein aggregation in biopharmaceutical formulation." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/effect-of-arginine-glutamate-on-protein-aggregation-in-biopharmaceutical-formulation(4ad30e3d-a3c2-4471-92cf-c1f029545044).html.
Full textThomas, Rhys David. "The role of protein dynamics in the aggregation of biopharmaceuticals." Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/15634/.
Full textCooper, Merideth A. "Creating an Efficient Biopharmaceutical Factory: Protein Expression and Purification Using a Self-Cleaving Split Intein." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu152226172238882.
Full textPenafuerte, Diaz Claudia. "IL2-based fusion proteins as new bi-functional biopharmaceuticals for the therapy of cancer." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103578.
Full textLa protéine de fusion comprenant le facteur stimulant les colonies des granulocytes-macrophages murins (GM-CSF) et l'interleukine-2 (IL2), GIFT2. GIFT2 possède de nouvelles propriétés immunologiques par rapport à l'utilisation des deux cytokines, telles que le recrutement d'un plus grand nombre de macrophages et de cellules NK dans un mélanome. Par conséquent, GIFT2 empêche la formation de tumeurs chez la souris implantée avec des cellules de mélanome B16. Dans le chapitre 2, j'ai évalué la capacité de GIFT2 à induire une réponse anti-tumorale in vivo contre des cellules B16 non-modifiées. J'ai remarqué que GIFT2 induit un effet spectateur sur les cellules B16 non-modifiées, et que cet effet est médié par les cellules NK. Toutefois, cet effet spectateur immunitaire se perd lorsque le nombre total de cellules B16 passe de 104 à 106. Avec ce plus grand nombre de cellules B16, j'ai observé une réduction substantielle du nombre de cellules NK infiltrants. J'ai déterminé que le facteur principal produit par la tumeur qui supprimait l'effet spectateur de GIFT2 est le TGF actif. J'ai observé que le TGF actif a diminué l'expression du récepteur de l'IL-2 (IL-2R) ainsi que la sécrétion de l'IFNg par les cellules NK. J'ai démontré que lorsque la sécrétion de TGF par les cellules B16 est inhibée, l'effet spectateur de GIFT2 est considérablement amélioré. Due aux propriétés immunostimulantes de la protéine de fusion GIFT2 murine, j'ai développé et évalué l'orthologue humain de GIFT2. Ceci est décris dans le chapitre 3. La GIFT2 humaine peut servir comme un moyen de générer des cellules NK oncolytiques pour la thérapie cellulaire du cancer. J'ai remarqué que la GIFT2 humaine induit une activation robuste de cellules NK ex vivo avec une sécrétion significative de cytokines/chemokines pro-inflammatoires et une expression augmentée de marqueurs d'activation de cellules NK. Ce phénotype est corrélé à une plus grande cytotoxicité contre les cellules tumorales. Au niveau moléculaire, la GIFT2 humaine mène à une activation puissante de la voie de signalisation Jak/STAT. Suivant ces résultats, j'ai proposé que l'inhibition du TGF actif pourrait améliorer l'immunothérapie génique d'un cancer existant. Dans le chapitre 4, je décris la production d'une nouvelle protéine de fusion entre l'IL-2 et le domaine extracellulaire du récepteur II de TGF (TGFRII soluble). Cette protéine chimérique appelé FIST a été générée pour contrarier les effets du TGF et aussi pour agir comme stimulus immunitaire pro-inflammatoire. J'ai observé que FIST agit comme récepteur du TGF actif qui se trouve en solution, et aussi interagit directement avec les cellules IL-2-sensibles. Ceci induit une hyperactivation de STAT1 en aval du récepteur de l'IL-2, ce qui donne lieu à une surexpression de SMAD7. De plus, l'hyperactivation de STAT1 induit une sécrétion significative de CXCL10, et augmente l'expression de T-bet et des gènes cibles de T-bet dans les cellules NK. Par conséquent, FIST empêche la formation de tumeurs non seulement chez la souris immunocompétente, mais aussi chez différentes souris immunodéficientes. Par contre, les souris avec fonction NK défectueuse, telles que les souris diabétiques non obèses présentant une immunodéficience combinée sévère (NOD-SCID) et les souris Rag2/c-/- ont développé des tumeurs. Tel que décrit dans le chapitre 5, j'ai générer et caractériser les cellules B stimulées par FIST. Les cellules B stimulées par la protéine FIST agissent comme des cellules présentatrices d'antigènes (APC) efficaces qui induisent l'activation et la prolifération de cellules T CD4+ et CD8+ antigène-spécifiques. Ce qui est aussi très intéressant est que les cellules B stimulées par FIST confèrent une immunité protectrice complète contre le "challenge" de la tumeur EG7. En conclusion, ces nouvelles protéines chimères bi-fonctionnelles sont des produits biopharmaceutiques potentiels pour le traitement du cancer.
Riley, Aidan. "Analysis and exploitation of GPI anchors in the expression of recombinant protein biopharmaceuticals." Thesis, University of Sheffield, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.578056.
Full textByun, Sangwon. "Transport of proteins, biopharmaceuticals and small pharmaceutical compounds into normal and injured cartilage by Sangwon Byun." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/60140.
Full text"June 2010." Cataloged from PDF version of thesis.
Includes bibliographical references.
Traumatic joint injury can induce acute damage to cartilage and surrounding joint tissues accompanied by an inflammatory response, which can significantly increase the risk of developing Qsteoarthritis. The mechanism by which joint injury results in disease development is not fully understood. However, chondrocyte metabolism is greatly affected by the transport properties of cartilage extracellular matrix, which determine the accessibility and the concentrations of various proteins and therapeutic agents to cells and cell receptors. Using in vitro models of mechanical injury to cartilage, we have characterized the uptake and binding of proteins and biopharmaceuticals in normal articular cartilage and have compared the results to those in cartilage subjected to mechanical injury and pro-inflammatory cytokines. We studied equilibrium partitioning and non-equilibrium transport into cartilage of Pfpep, a 760 Da positively charged peptide inhibitor of the pro protein convertase PACE4. Competitive binding measurements revealed negligible binding to sites in the matrix. The uptake of Pf-pep depended on GAG charge density, consistent with predictions of Donnan equilibrium. The diffusivity of Pf-pep was measured to be ~1 x 10-6 cm2/s, close to other similarly-sized non-binding solutes. These results suggest that small positively charged therapeutics will have a higher concentration within cartilage than in the surrounding synovial fluid, a desired property for local delivery; however, such therapeutics may rapidly diffuse out of cartilage unless there is additional specific binding to intratissue substrates that can maintain enhanced intratissue concentration. We have also examined the effect of mechanical injury and inflammatory cytokines, TNFa, on the uptake of anti-IL-6 antibody Fab fragment (48 kDa). Anti-IL-6 Fab was able to penetrate into cartilage, though final equilibrium uptake would likely occur only after 6-10 days within 1 mm thick explant disks. Uptake of anti-IL-6 Fab was significantly increased following mechanical injury of the cartilage in vitro. A further increase in uptake was caused by TNFa treatment combined with mechanical injury. The increase in uptake was accompanied by GAG loss from the tissue, suggesting that there can be greater accessibility of large solutes into cartilage after direct mechanical injury or inflammatory cytokine treatment to the tissue, where the increase in uptake was related with the severity of matrix damage and loss. We also studied the binding and uptake of TNFa in articular cartilage and observed significant binding of TNFa to matrix sites. Binding was stronger for the monomeric form of TNFa compared to trimeric form. Binding of TNFa was not disrupted by pre-treatment of the tissue with trypsin, indicating that the intra-tissue binding sites were not removed by trypsininduced proteolysis of the matirx. These results suggest that matrix binding as well as monomer-trimer conversion of TNFa both play crucial roles in regulating the accessibility of TNFa to cell receptors. The results of this thesis are significant in that they suggest that injurious mechanical loading and inflammatory cytokine applied to cartilage can affect transport processes within the tissue. The resulting altered transport, in turn, can influence the accessibility of proinflammatory cytokines and anti-catabolic drugs which are designed to treat pathogenesis of OA.
Ph.D.
Zhai, Yujing. "Studies of Split Intein-Mediated Self-Cleaving Tag for Protein Purification." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480517677272206.
Full textRodrigues, Mariane Augusta Domingues. "Avaliação da atividade e resistência à clivagem proteolítica de L-asparaginases recombinantes obtidas por reação em cadeia da polimerase propensa a erro." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/9/9134/tde-24082016-163433/.
Full textEscherichia coli L-asparaginase (EcA II) is an enzyme widely used in the treatment of acute lymphoblastic leukemia (ALL), acting in the depletion of the amino acid L-asparagine, which is essential for cancer cells proliferation. However, treatment with L-asparaginase is associated with a high rate of hypersensitivity, due to formation of anti-L-asparaginase antibody and the enzyme cleavage by the serum proteases asparagine endopeptidase (AEP) and cathepsin B (CTSB). Furthermore, the neurotoxicity is associated with the effect of the enzyme L-glutaminase activity. The main aim of the current work is to obtain variants of EcA II (gene ansB) with an equivalent catalytic efficiency, greater resistance to proteolytic cleavage and a reduced glutaminase activity. For such purpose, through error-prone polymerase chain reaction (epPCR) of gene ansB, a library of 1128 clones was constructed in pET15b vector and expressed in BL21(DE3). None mutant with an asparaginase activity equivalent to EcA II wild type showed a reduced glutaminase activity. Among the screened clones, one mutant (T161I) was resistant to CTSB proteolytic cleavage and two mutants (Q190L e P40S/S206C) were resistant to both CTSB and AEP proteolytic cleavages. These three mutants were EcA II wild type equivalents in asparaginase and glutaminase activities. Our data show promising new possibilities of mutant EcA II presenting higher stability against human serum proteolytic cleavage and maybe lower immunogenicity.
Husson, Gauthier. "Development of host cell protein impurities quantification methods by mass spectrometry to control the quality of biopharmaceuticals." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAF066/document.
Full textRecent instrumental developments in mass spectrometry, notably in terms of scan speed and resolution, allowed the emergence of “data independent acquisition” (DIA) approach. This approach promises to combine the strengths of both shotgun and targeted proteomics, but today DIA data analysis remains challenging. The objective of my PhD was to develop innovative mass spectrometry approaches, and in particular to improve DIA data analysis. Moreover, we developed an original Top 3-ID-DIA approach, allowing both a global profiling of host cell proteins (HCP) and an absolute quantification of key HCP in monoclonal antibodies samples, within a single analysis. This method is ready to be transferred to industry, and could provide a real time support for mAb manufacturing process development, as well as for product purity assessment
Engström, Mathias, Olle Pontén, Carlsson Philip, Nadeen Bahnam, Ella Strömberg, and Oskar Westlin. "Antibiotic free and optimised protein production using Escherichia coli." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-352528.
Full textDe, 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 textENGLISH 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.
Liste, Calleja Leticia. "Study and characterisation of human HEK293 cell line as a platform for recombinant protein production." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/308324.
Full textThe thesis is focused on the study of recombinant protein production in mammalian cell lines. In particular, the study of three different approaches of different bioprocesses based on HEK293 cells has been addressed. As a model protein for recombinant expression, CapPCV2 has been selected. This protein makes up the viral capsid of Porcine circovirus serotype 2 (PCV2), which is the causative agent of PCVDs (porcine circovirus diseases), a group of diseases with major impact in pig’s industry worldwide. This project has been addressed from the perspective of bioprocess development and optimization and therefore, the increment of volumetric production of cells, virus and proteins have been the driving force of the research. Firstly, cell culture media and nutritional supplementation studies are presented. Cell growth relies in high extent to the nutritional and physicochemical characteristics of the media in which cells are cultured and therefore, finding the proper cell media is one of the key factors for cell culture expansion. The initial media study resulted in a 6-‐fold increment of the maximal viable cell achieved in the original media. Besides, different cell culture strategies have been explored, which resulted in a fed-‐batch strategy that allowed reaching maximal viable cell densities of 26.8x106 cell/mL, which represents 13-‐fold increment on maximal viable cell density originally reached. In the second and third chapter of results, three different approaches for the expression of recombinant CapPCV2 (r-‐CapPCV2) are evaluated and discussed. As a first approach, a viral recombinant adenovirus encoding for the gene CapPCV2 has been generated and used as viral vector for the production of the recombinant protein in HEK293 cells. Besides, a deep study of the main parameters that affect the infection performance has been carried out and discussed in order to find the best media, MOI (multiplicity of infection), TOI (time of infection) and TOH (time of harvest) for adenovirus and recombinant protein production. This study was performed with an adenovirus expressing the reporter gene GFP and thereafter, the best infection parameters encountered were applied for the production of r-‐CapPCV2 (media: SFMTransFx-‐293 supplemented with 4mM glutaMAX, 5% FBS and 10%CB5; MOI:1; TOI:1x106 cell/mL) and TOH:48hpi). The second and third strategies are both based on the generation of stable producer cell lines, but one strategy relies on illegitimate (or random) integration of the gene in the HEK293 genome ,whereas the other strategy is a site-‐directed integration of the gene in previously characterized hot-‐spots (i.e. high-‐active transcribed regions from genome). The site-‐directed integration was performed using RMCE technology (Recombinant mediated cassette exchange). After the comparison of the specific and volumetric productivities achieved with each approach, the best producer has been selected. Nevertheless, r-‐CapPCV2 was poorly produced so it was unfeasible to develop/design a cost-‐effective industrial bioprocess and other alternatives must be studied in the future. Finally, the study of an unexpected metabolic behaviour observed in HEK293 cells cultured in our lab has been addressed from a physiologic and metabolic perspective. HEK293 cells could concomitantly consume glucose and lactate in exponentially growing cultures at particular environmental conditions. After a deep study of these conditions, it was found out that the switch from lactate secretion (which is the main drawback of mammalian high cell density cultures) to lactate consumption can be triggered from the beginning of cell culture at pH0=6.6 together with the addition of 4-‐12mM of lactate to media. Remarkably, under these conditions nor cell growth neither protein production were negatively affected. Form these results, we hypothesize that HEK293 can co-‐transport lactate and H+ to the cytosol as a pH-‐detoxification mechanism. Moreover, the application of flux balance analysis permitted to find out that when lactate and glucose are consumed together a “more balanced” metabolism is achieved, meaning that glycolytic and TCA fluxes became similar, avoiding pyruvate accumulation at the cytosol and consequently, lactate formation. This is totally opposed to the extensively observed metabolism of exponentially growing mammalian cell lines, where the high flux through the glycolytic pathway encounters a limitation on the fluxes entering the mitochondria (hence, the TCA cycle) and consequently lactate is produced and secreted to media. The construction of a HEK293 metabolic model and the application of FBA will allow making in silico predictions of metabolic beahaviours after the upregulation or downregulation of target genes. This strategy may open the possibility of generate engineered HEK293 cell lines with an optimised metabolism in order to study more efficient cell culture strategies towards the achievement of higher cell densities and product titres.
Custodio, Débora Fernandes. "Caracterização de L-Asparaginase de Erwinia chrysanthemi melhorada por evolução sintética de proteínas e otimização das condições de produção." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/9/9134/tde-11062018-104646/.
Full textL-Asparaginase (L-ASNase) is a bacterial tetrameric enzyme used in chemotherapy sessions that deplete asparagine (Asn) and glutamine (Gln), transforming them into Aspartate (Asp) or glutamate (Glu), respectively, and ammonia. However, L-ASNase can induce immune response leading to the production of anti-asparaginase antibody, an important cause of drug resistance. Ideally, L-ASNase would be one with high activity, high stability and low immunogenic potential, but the L-ASNases commercially available today do not present these characteristics simultaneously. For this reason, this study used techniques of random and site-directed mutagenesis in order to create a new proteoform of E. chrysanthemi L-ASNase with improved activity and stability. In addition, culture conditions were studied in a metabolic shaker, aiming at the optimization of production conditions. A library with 1,056 clones was created, and of these clones, 19 were selected because they had activity superior or equal to the wild-type enzyme in crude protein extract. Among them, 2 mutants stood out for having different glutaminase specific activity in relation to wild-type enzyme. The 9-6D mutant also showed decreased structural disorder and immunogenic epitopes. The 9-5F mutant demonstrated a decrease in percentage of glutaminase activity when compared to the wild-type enzyme. The production study of 9-5F mutant indicated that the induction temperature followed by the inductor concentration are the most relevant parameters for the production optimization of E. chrysanthemi mutant L-ASNase.
Pow, Andrew James. "Protein complementation assay as a display system for screening protein libraries in the intracellular environment." Thesis, Queensland University of Technology, 2008. https://eprints.qut.edu.au/30392/1/Andrew_Pow_Thesis.pdf.
Full textPow, Andrew James. "Protein complementation assay as a display system for screening protein libraries in the intracellular environment." Queensland University of Technology, 2008. http://eprints.qut.edu.au/30392/.
Full textPetri, Niclas. "Involvement of Membrane Transport Proteins in Intestinal Absorption and Hepatic Disposition of Drugs Using Fexofenadine as a Model Drug." Doctoral thesis, Uppsala University, Department of Pharmacy, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5808.
Full textThe aims of this thesis were to study the in vivo relevance of membrane transporters for intestinal absorption and the hepatic disposition of drugs in humans and preclinical models. Fexofenadine is a substrate for ABCB1 (P-glycoprotein) and members of the organic anion transporting polypeptide (OATP/SLCO) family. It is marginally metabolised in humans.
The influence of known inhibitors of ABCB1 and OATPs on the membrane transport and pharmacokinetics of fexofenadine was investigated in Caco-2 and porcine models and in humans. The permeability of fexofenadine remained low, even when significantly altered by the addition of an inhibitor. Using the Loc-I-Gut® technique in vivo in humans, it was possible to see that the jejunal effective permeability of fexofenadine was unchanged when given with verapamil. However, the systemic exposure and apparent absorption rate of fexofenadine increased. This suggests that the first-pass liver extraction of fexofenadine was reduced by verapamil, probably through the inhibition of sinusoidal OATP-mediated and/or canalicular ABCB1-mediated secretion. The unchanged permeability can be explained by simultaneous inhibition of jejunal apical OATP-uptake and ABCB1-efflux, which would leave fexofenadine to be transported by passive trancellular diffusion. A Loc-I-Gut® perfusion in the porcine model enabling blood sampling in the portal and hepatic veins and bile collection revealed increased jejunal permeability, but no subsequent verapamil-induced elevation in the systemic exposure of fexofenadine. This indicates a species-related difference in the localisation of and/or the substrate specificity of fexofenadine for the transporters involved. The absence of an effect on the first-pass liver extraction in the porcine model might be caused by the observed lower liver exposure of verapamil.
Finally, a novel intubation technique enabling dosing of fexofenadine in the jejunum, ileum and the colon showed that fexofenadine was absorbed less along the length the intestine in agreement with the properties of a low permeability drug.
West, Nathan R. "Development of a tunable mammalian protein expression system and an investigation of promoter interference in three promoters often utilized in the production of biopharmaceuticals." Thesis, University of Sheffield, 2014. http://etheses.whiterose.ac.uk/6701/.
Full textKohli, Neha. "Amelioration of Amyloid Burden in Advanced Human and Mouse Alzheimer's Disease Brains by Oral Delivery of Myelin Basic Protein Bioencapsulated in Plant Cells." Master's thesis, University of Central Florida, 2012. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/5380.
Full textM.S.
Masters
Molecular Biology and Microbiology
Medicine
Biotechnology
Vaidilaite-Pretorius, Agita. "Mechanistic approaches towards understanding particle formation in biopharmaceutical formations : the role of sufactant type and level on protein conformational stability, as assessed by calorimetry, and on protein size stability as assessed by dynamic light scattering, micro flow imaging and HIAC." Thesis, University of Bradford, 2013. http://hdl.handle.net/10454/13482.
Full textSilva, Bruna Daniela Gonçalves da. "Nanopartículas lipídicas para a administração de produtos biofarmacêuticos." Master's thesis, [s.n.], 2015. http://hdl.handle.net/10284/5187.
Full textOs produtos biofarmacêuticos englobam os produtos à base de proteínas terapêuticas, de ácidos nucleicos e os que são usados em terapia celular. Com o crescimento exponencial que se tem verificado nos últimos anos para a biotecnologia farmacêutica, o uso clínico destes produtos tem vindo a aumentar. Contudo, tendo em conta as características físico-químicas das moléculas, têm surgido algumas limitações relativas à administração de produtos biofarmacêuticos. Com efeito, a investigação tem-se focado nos estudos relativos ao desenvolvimento de novos sistemas para veicular estes produtos. Neste contexto, e tendo em conta as vantagens que apresentam, as nanopartículas lipídicas têm sido apresentadas como promissoras. Na primeira parte deste trabalho é efectuada uma revisão bibliográfica relativa aos diferentes produtos biofarmacêuticos, às suas características e limitações de administração. Na segunda parte, é apresentada uma revisão relativa ao estado da arte do uso de nanopartículas lipídicas para promover a administração de produtos biofarmacêuticos. Biopharmaceutical products include therapeutic proteins, nucleic acids and cell-based products. Within the exponential growth of pharmaceutical biotechnology the clinical use of these products has been increasing. Nevertheless, according to the physical and chemical nature of the molecules, some limitations have appeared, limiting the use of biopharmaceutical products. In this way, the number of studies related with the development of new solutions for using biopharmaceutical products has been growing. Accordingly, due to its advantages, lipid nanoparticles have been presented as promising candidates. The first part of this work provides a bibliographic overview of the different biopharmaceutical products, and their characteristics and limitations for therapeutic use. In the second part, is presented a review of the state-of-the-art of using lipid nanoparticles systems to improve the therapeutic efficacy of biopharmaceutical products.
Jinzenji, Daniela. "Desenvolvimento de processo cromatográfico para purificação de fator VIII humano. Emprego de anticorpos contra fragmentos específicos da proteína na avaliação da pureza e estabilidade durante as etapas de purificação." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-10032009-104314/.
Full textCoagulation factor VIII (FVIII), recombinant or purified from plasma, is the biopharmaceutical used for treatment of haemophilia A, the most frequent human hemorrhagic disorder. The traditional method used for purification of FVIII starts from plasma cryoprecipitate and alcoholic precipitation. The Instituto Butantan proposed an alternative methodology using only chromatography for FVIII purification. The main objective of this project was to compare two chromatographic methods for FVIII purification: 1 - direct plasma gel filtration and 2 - pre-purification of FVIII by anion exchange chromatography, followed by gel filtration. The purification process was analyzed by determination of FVIII specific activity and detection of other coagulation factors co eluting in chromatographic fractions. Fragments of FVIII gene were cloned and protein fragments were expressed for animal immunization. Sera with polyclonal antibodies anti-FVIII were used in western blots assays to detect FVIII chains or its degradation.
Bright, Andrew G. "Mechanistic Insights into the Stabilisation of Biopharmaceuticals using Glycine Derivatives. The Effect of Glycine Derivatives on the Crystallisation, Physical Properties and Behaviour of Commonly used Excipients to Stabilise Antigens, Adjuvants and Proteins in the Solid State." Thesis, University of Bradford, 2015. http://hdl.handle.net/10454/15943.
Full textSunley, Kevin. "The Adaptation of Chinese Hamster Ovary Cells to Hypothermic Temperatures Increases Yields of Monomeric Recombinant Interferon-beta." 2009. http://hdl.handle.net/1993/3192.
Full textCharlton, Adam. "Improved production of biopharmaceuticals by site-specific cleavage of fusion proteins expressed in Escherichia coli." 2008. http://hdl.handle.net/2440/59637.
Full textThe recombinant expression of heterologous proteins in microorganisms, such Escherichia coli, is often improved by producing the protein of interest translationally linked to another, often unrelated, protein giving rise to a "fusion protein" construct. For many applications it is desirable or imperative to separate the extraneous material from the protein of interest. An increasingly popular approach to this task is the use of site-specific endoproteases 10 excise the protein product. A number of commercially available site-specific proteases exist, but many are not capable of generating an authentic N-terminus for the product, display unsatisfactory specificity leading to adventitious cleavage of the product, or they are unsuitable for an industrial process. Mutants of the serine protease a-Lytic protease have been shown to satisfy many of the criteria for an industrially suitable protease and have been applied to the cleavage of some important fusion proteins used in the production of members of the Insulin-like Growth Factor (IGF) family Lacking from these examples, however, is any viable proteolytic solution for the liberation of human IGF-I from fusion proteins. This has been primarily attributed to the Proline bearing N-terminal tripeptide sequence of this protein, which is known to be refractory to the activity of many site-specific proteases. It has been suggested that in the generation of two combinatorial mutant libraries of a-Lytic protease, the preference for amino acids C-terminal to the cleavage site may have been altered. It is the purpose of this work to first determine if such an alteration has been made in any of the mutants so as to allow cleavage immediately before the N-terminus of human IGF-1, and then to task the lead mutant(s) to the cleavage of the full-length fusion protein. All members of the two mutant libraries were cultured and their activity confirmed and quantified against a generic B-casein substrate in a high-throughput assay. A second high-throughput technique was then employed to query the mutant proteases for their ability to catalyse proteolysis at the required sequence in a peptide model. Finding that many mutants appeared successful at this task, the findings were verified on a longer peptide model of the cleavage site. Initially the yields achieved by cleavage of the full-length IGF-1 fusion protein by a lead candidate mutant a-Lytic protease were not sufficient to satisfy the requirements of an industrial process, despite alteration of the reaction conditions. However, the insight gained from these reactions could be applied to the redesign of the protein structure around the intended site of cleavage, significantly improving site-specific proteolysis. The IGF-1 generated by this cleavage has been shown to be bioequivalent to commercial reference standard to cultured mammalian cells and the yield of this process is approximately 5-fold improved over the existing cleavage system.
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Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008
Charlton, Adam. "Improved production of biopharmaceuticals by site-specific cleavage of fusion proteins expressed in Escherichia coli." Thesis, 2008. http://hdl.handle.net/2440/59637.
Full textThe recombinant expression of heterologous proteins in microorganisms, such Escherichia coli, is often improved by producing the protein of interest translationally linked to another, often unrelated, protein giving rise to a "fusion protein" construct. For many applications it is desirable or imperative to separate the extraneous material from the protein of interest. An increasingly popular approach to this task is the use of site-specific endoproteases 10 excise the protein product. A number of commercially available site-specific proteases exist, but many are not capable of generating an authentic N-terminus for the product, display unsatisfactory specificity leading to adventitious cleavage of the product, or they are unsuitable for an industrial process. Mutants of the serine protease a-Lytic protease have been shown to satisfy many of the criteria for an industrially suitable protease and have been applied to the cleavage of some important fusion proteins used in the production of members of the Insulin-like Growth Factor (IGF) family Lacking from these examples, however, is any viable proteolytic solution for the liberation of human IGF-I from fusion proteins. This has been primarily attributed to the Proline bearing N-terminal tripeptide sequence of this protein, which is known to be refractory to the activity of many site-specific proteases. It has been suggested that in the generation of two combinatorial mutant libraries of a-Lytic protease, the preference for amino acids C-terminal to the cleavage site may have been altered. It is the purpose of this work to first determine if such an alteration has been made in any of the mutants so as to allow cleavage immediately before the N-terminus of human IGF-1, and then to task the lead mutant(s) to the cleavage of the full-length fusion protein. All members of the two mutant libraries were cultured and their activity confirmed and quantified against a generic B-casein substrate in a high-throughput assay. A second high-throughput technique was then employed to query the mutant proteases for their ability to catalyse proteolysis at the required sequence in a peptide model. Finding that many mutants appeared successful at this task, the findings were verified on a longer peptide model of the cleavage site. Initially the yields achieved by cleavage of the full-length IGF-1 fusion protein by a lead candidate mutant a-Lytic protease were not sufficient to satisfy the requirements of an industrial process, despite alteration of the reaction conditions. However, the insight gained from these reactions could be applied to the redesign of the protein structure around the intended site of cleavage, significantly improving site-specific proteolysis. The IGF-1 generated by this cleavage has been shown to be bioequivalent to commercial reference standard to cultured mammalian cells and the yield of this process is approximately 5-fold improved over the existing cleavage system.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008
"Optimization of a Viral System to Produce Vaccines and other Biopharmaceuticals in Plants." Doctoral diss., 2017. http://hdl.handle.net/2286/R.I.46337.
Full textDissertation/Thesis
Doctoral Dissertation Microbiology 2017
Das, Anindita. "Nanoparticle Mediated Suppression of Protein Aggregation." Thesis, 2015. http://etd.iisc.ernet.in/2005/3521.
Full text