Dissertations / Theses: 'Biopharmaceutical proteins'

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Wei, Tzu-Hsiang Biotechnology &amp 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.

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The creation of stable mammalian cell lines for biopharmaceutical production often require several months, and is unfavourable for the rapid production of multiple drug candidates for screening in the early stages of development. Biopharmaceutical production by transient transfection provides a possible alternative of quickly producing these early stage drug candidates. The Epi-CHO transient expression system, which consists of a Chinese hamster ovary (CHO) cell line (CHO-T) expressing the murine polyomavirus Large T-Antigen (LT), emonstrated enhanced transient recombinant protein production. The aim of this study was to prolong transient recombinant protein prod.Jction of the Epi-CHO expression system by creating a CHO cell line expressing both LT and EBNA1 (ECHO-T). The pEBNA1-LT expression vector encoding LT and EBNA1 was constructed and transfected into CHO-K1. A total of 20 clones were isolated from the antibioticresistant pool and screened for the expression of functional LT and EBNA1. PCR analysis showed 16 of the 20 clones was positive for EBNA1 and LT DNA. Of the 16 clones, six were positive for EBNA1 and LT expression by RT-PCR. Detection of LT and EBNA1 by immunofluorescence showed positive staining for the P7-G3 clone. Western blotting suggested the P7-G3 clone was: positive for EBNA1, and clones P3-C7 and P7-E2 were positive for LT. A plasmid replication assay confirmed the expression of functional LT in all six clones. Plasmid maintenance assay confirmed clone P7-G3 as the ECHO-T clones to express functional EBNA1. The P7-G3 clone demonstrated prolonged and sustained transient recombinant protein expression when compared to CHO-T. The P7-G3 clone achieved sustained transient protein expression for 32 days in the absence of selection, the longest currently reported for CHO cells.

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Pradhan, 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/.

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Enterokinase (EK) is a serine protease used to cleave therapeutic recombinant proteins during downstream processing. It has been selected for the activation and cleavage of a range of proprietary fusion proteins developed by Syntaxin Ltd. Whilst EK is well suited to this role in regards to substrate specificity, it has drawbacks, especially when it comes to expression in bacterial cells. Expression of EK in bacterial cells is the preferred expression method for process optimisation but is problematic due to its preference for inclusion body formation. This project describes efforts for improving the solubility of EK in E. coli using different constructs and mutagenesis. A total of four constructs were tested with two found to be soluble and one partially soluble. Two of the constructs (D4K-EK & pelB-EK) were found to readily form inclusion bodies (IB). Refolding of these constructs was undertaken and optimised using DoE. Only the refolded pelB EK showed significant activity, but refolded activity was found to vary greatly based on IB quality. The partially soluble pelB-EK construct exports to the periplasm for activation and soluble expression and was chosen for mutagenesis studies to improve soluble expression. A rational design approach using a range of sequence and structural bioinformatics methods including the consensus sequence, Hotpatch and statistical coupling analysis were utilised to identify fifteen stabilising mutants and seven mutants designed to increase surface charge. Of these potential mutants, ten (five stabilising, five surface charge) were created and analysed for activity, soluble yield and changes to secondary structure. Seven of the ten mutants showed measurable activity. Of interest were the surface charge mutants, which helped improve the purified yield by up to 2.5 fold. Also of note was consensus mutant V30Q which helped improve activity of periplasmic EK by 4.3 fold, whilst A32S and A44G visibly improved the thermo-tolerance of EK.

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Batra, Sumit. "Innovative Purification Protocol for Heparin Binding Proteins: Relevance in Biopharmaceutical and Biomedical Applications." TopSCHOLAR®, 2011. http://digitalcommons.wku.edu/theses/1062.

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Heparin binding (HB) proteins mediates a wide range of important cellular processes, which makes this class of proteins biopharmaceutically important. Engineering HB proteins could bring many advantages, but it necessitates cost effective and efficient purification methodologies compared to the currently available methods. One of the most important classes of heparin binding protein is the fibroblast growth factors (FGFs) and its receptors (FGFRs). In this study, we report an efficient off-column purification of FGF-1 from soluble fractions and purification of the D2 domain of FGFR from insoluble inclusion bodies, using a weak amberlite cation (IRC) exchanger. This approach is an alternative to conventional affinity column chromatography, which exhibit several disadvantages, including time-consuming experimental procedures and regeneration and results in high cost for production of recombinant proteins. Authenticity of the purified proteins was verified by SDS-PAGE and MALDI mass spectrum analysis. Results of the heparin binding chromatography and steady state fluorescence experiments showed that the FGF-1 and the D2 are in a native biologically active conformation. The findings of this study will not only aid an in-depth investigation of this class of proteins but will also provide avenues for inexpensive and efficient purification of other important biological macromolecules.

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Buyel, 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.

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Hameed, 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.

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Aqueous Phase Partitioning has a long history of applications to the analytical characterisation of biomolecules. However process applications have attracted the most interest in biotechnology where it has become widely recognized as a cost-effective technique. The main aim of this work was to explore the proposition that partition in Aqueous Two Phase Systems (ATPS) can be used as an analytical tool to detect protein isoforms and to assess the applicability of the method in clinical assays and for quality control in bioprocessing through examination of several analytical problems. The work also examined the development of automated methods of system preparation and sampling techniques to determine the partition coefficient in ATPS. The study demonstrated that the geometrical form of the phase diagram co-existence curve was of crucial importance since this directly affected the accuracy with which systems of defined Tie Line Length and Mass Ratio could be constructed. The TLL %Bias (accuracy) of a theoretical system range in the PEG1000-(NH4)2SO4 system at shorter TLL (12.2) was in the range +80.6% to -100% while at a longer TLL (53.1) the %Bias (accuracy) was reduced to +0.1% to -1.9%. At the same time the MR %Bias (accuracy) at shorter TLL (12.2) was in the range +59.5% to -21.3% while at the longer TLL (53.1) this was reduced to +2.7% to -2.6%. By contrast in the PEG8000-Dextran500 system the TLL %Bias (accuracy) at shorter TLL (13.1) was in the range +3.7% to -4.12%, while at a longer TLL (31.1) the range was +0.74% to -0.67%. The MR %Bias (accuracy) at the shorter TLL (13.1) was in the range +3.6% to -3% while at the longer TLL (31.1) the range was +1.1% to -1.4%. This illustrated that it is more difficult to work with a high degree of accuracy (e.g. %Bias <5%) close to the critical point in PEG-salt systems than in PEG-dextran systems. Two different approaches were taken to examine analytical phase partitioning. In the first approach the structure of the isoforms of a model protein (ovalbumin) were altered enzymatically. Analytical methods involving Strong Anion-Exchange chromatography were developed and applied to the separation of the ovalbumin isoforms. Removal of the phosphorylated groups (dephosphorylation of ovalbumin) was undertaken using alkaline phosphatase and de-glycosylation was attempted using neuraminidase and Endo-glycosidase F. However, both enzymatic approaches to deglycosylation were unsuccessful. Dephosphorylated isoforms were successfully produced and characterised. After partitioning in ATPS a clear difference was demonstrated between the behaviour of the native and dephosphorylated forms of ovalbumin. The mean % recovery in a PEG-salt ATPS was 99.8% (± 3.59) for the naive protein and 75.6% (± 4.03) for the dephosphorylated form. On the other hand, in a PEG3350-Dextran500 system, where solubility was maintained, a significant difference in the partition coefficient (K) of native and dephosphorylated ovalbumin was found. K for native ovalbumin was 0.85 while the partition coefficient of the dephosphorylated ovalbumin was 0.61. Analysis of covariance (ANCOVA) indicated that the regression coefficients of the respective partition isotherms were significantly different (p value < 0.05). In a second approach to examine analytical phase partitioning, chemical modification of a specific target surface amino acid of another model protein (serum albumin) was used to determine the degree of conjugation of the protein and also to determine its oxidative state. The method examined the reactivity of a free surface thiol to a wide range of labels ( (a) 2-methylsulfonyl-5-phenyl -1,3,4 oxidiazole reagent, (b) N-Ethylmaleimide (NEM) reagent, (c) 5, 5’-dithiobis (2-nitrobenzoate)(DTNB) (Ellman’s reagent), (d) N-pyrenylmaleimide (NPM) reagent, (e) Fluorescein-5-maleimide (F-5-M) Reagent). Only DTNB was found to modify the surface free thiol of serum albumin in a highly specific and quantitative manner. In the course of the development of a partitioning assay for surface free thiols of serum albumin significant oxidative properties were found to be associated with poly(ethylene glycol) PEG solutions and several attempts were made to find an oxidatively safe partitioning system by including antioxidants and by removal of contaminants by freeze drying. PEG3350-Dextran500 was found to provide an oxidatively safe environment for the development of a partitioning assay for the determination of albumin free thiols. A phase partitioning assay system capable of quantitatively resolving protein associated free thiols and low molecular weight thiols from a mixture of the two was developed. Correlation coefficients (R2) for the regression of experimentally determined protein free thiols in the presence of different levels of added LMW free thiol on the known addition of protein ranged from 0.77 to 0.83. The results demonstrated that the assay could quantify and distinguish both types of thiol in a simple two-step procedure.

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Buyel, 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.

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Dehling, 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.

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Dehling, 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.

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Arfi, 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.

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Arfi, 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.

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Hebditch, 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.

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Antibodies and antibody fragments are the largest class of biotherapeutics in development with many products already available in the clinic. Antibodies are promising due to their naturally high affinity and specificity for biological targets. A key stumbling block to biopharmaceutical development compared to small molecule drugs is the general requirement for a stable liquid formulation, which is often difficult to obtain due to issues with aggregation, phase separation, particle formation, and chemical instabilities. Aberrant solution behaviour limits the production, storage and delivery of the monoclonal antibody. Biopharmaceutical solution behaviour is determined by weak, transient protein-protein and protein-solvent interactions. An attractive interaction potential between proteins in solution can lead to association. Irreversible association occurs when proteins undergo large scale structural changes and aggregate. Reversible association is less severe, but can lead to undesirable solution properties such as high viscosity, phase separation and opalescence, which can lead to difficulties throughout the downstream processing and formulation steps. These problems can become exacerbated during formulation of antibodies when trying to achieve high protein concentrations often required for effective antibody dosage. Firstly, we studied the domains of the Fab fragment using statistical models and continuum electrostatic calculations and found that the CH1 domain is more soluble than the other domains and has properties of intrinsically disordered like proteins which is supported by observations in the literature. We then investigated the immunoglobulin superfamily and found 11 proteins which may have a similarly disordered nature. We present a new web server for predicting protein solubility from primary sequence using an in-house algorithm that weighs the contribution of various sequence properties for predicting solubility. Lastly, we conducted physical characterisation of an antibody and human serum albumin in pharmaceutically relevant buffers and found that the interaction potential can be modelled using spherical models from low to high protein concentration. We hope that the work outlined in this thesis will contribute to the theoretical understanding and modelling of protein solution behaviour.

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McGarvey, 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.

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Fung, 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.

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Kheddo, 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.

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Monoclonal antibodies (mAbs) represent one of the fastest growing classes of therapeutic proteins. This success is due to a number of attractive properties such as high binding affinity, specificity, low immunogenicity and high aqueous solubility. Despite this, mAbs can suffer from undesirable physical instabilities, especially reversible self-association (RSA), which can lead to aggregation and phase separation. One aspect of formulation is therefore to find solution conditions which minimise mAb aggregation propensity during storage at high concentrations. Hence, the buffer, excipient and pH must be carefully considered to obtain the optimal formulation. Currently, if a platform formulation process is non-ideal for a particular candidate mAb, then an alternative strategy is to utilise high-throughput screening to measure various physical parameters indicative of physical stability. Arginine (in the form of hydrochloride salt Arg·HCl) is often used in formulations exhibiting high RSA and a propensity for aggregation. The interaction of Arg with the protein surface is complex and dependent on both the salt form and concentration. Here the focus was on the glutamate salt of arginine (Arg·Glu), to quantify its effect on mAb conformational and colloidal stability under different pH conditions. Arg·Glu was able to decrease the propensity of the mAbs to aggregate, particularly at pH values closer to their pI.The work also included the use of in vitro cell culture models to examine cell viability in the presence of the various arginine salts over a range of osmolalities. Whilst Arg·Glu is composed of two naturally occurring amino acids and both of which are considered non-toxic individually, the effect of the increased concentrations of their combination, on cells has not been explored previously. In vitro cell lines were chosen to represent the subcutaneous tissue, the effect of Arg·Glu on cell viability was compared against NaCl, Arg·HCl and sodium glutamate (NaGlu). The work concluded there was no additional toxicity associated with the presence of Arg·Glu in the cell culture models studied, therefore Arg·Glu has the potential as an excipient as it reduces aggregation and is nontoxic. Another aspect of the work was to assess the use of solution NMR spectroscopy as an orthogonal technique in mAb formulation characterisation. 1H NMR spectroscopy was used to measure a number of experimental parameters for high concentration mAb solution. The work proposed that 1H NMR spectroscopy can serve as a valuable orthogonal method for mAb characterization and formulation.

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Thomas, Rhys David. "The role of protein dynamics in the aggregation of biopharmaceuticals." Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/15634/.

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Cooper, 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.

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Penafuerte, 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.

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The murine fusion protein between Granulocyte macrophage Colony Stimulating Factor (GM-CSF) and Interleukin-2 (IL2, aka GIFT2) display novel immunological properties compared to both cytokines in combination such as greater melanoma site recruitment of macrophages and functional NK cells. Consequently, GIFT2 prevent tumor formation in mice implanted with genetically modified B16 melanoma cells. In the Chapter 2 of my thesis, I evaluated the bystander effect of the murine GIFT2 in vivo to induce an effective antitumor response against non-genetically modified B16 cells present in the tumor site. GIFT2 bystander effect on non modified B16 cells was mediated by recruited NK cells in the tumor site. However, the immune bystander effect was completely lost as tumor burden increase, which correlated with a sharp reduction in the number of tumor-infiltrating NK cells. I identified that active TGF is the main tumor-derived factor that downregulated IL-2R expression and IFNg secretion by NK cells, and therefore attenuated GIFT2-dependent bystander effect. We demonstrated that in vivo blockade of B16-derived TGF significantly improved the immune bystander effect arising from GIFT2. Based on the potent immunostimulatory properties of the murine GIFT2 on NK cells, in the chapter 3 I developed, characterized and evaluated the human ortholog of GIFT2, which may serve as a mean to generate oncolytic NK cells for cell-based therapy of cancer. The human GIFT2 induces robust NK cell activation ex vivo with significant secretion of pro-inflammatory cytokines, chemokines and upregulate the expression of activation markers and receptors on NK cells. This phenotype correlates with significantly greater cytotoxicity against tumor cells. At the molecular level, the human GIFT2 leads to a potent activation of Jak/STAT signaling pathway downstream of IL-2 receptor. In conclusion, hGIFT2 fusokine possesses unique biochemical properties and constitutes a novel and potent tool for ex vivo NK cell activation and maturation. Based on our results, cancer gene immunotherapy of pre-established tumors will be enhanced by blockade of active TGF. To antagonize TGF dependent effects in tandem with a pro-inflammatory immune stimulus, I generate of a new chimeric protein borne of the fusion of IL-2 and the soluble extracellular domain of TGF-receptor II (aka FIST). FIST acts as a decoy receptor trapping active TGF-in solution and directly interacts with IL-2-responsive cells, inducing a distinctive hyperactivation of STAT1 downstream of IL-2 receptor, which in turn promotes SMAD7 overexpression. STAT1 hyperactivation further induces significant secretion of CXCL10, upregulates T-Bet and T-Bet target gene expression in NK cells. The synergism of TGFblockade coupled to IL-2(R)-dependent STAT1 hyperagonism leads to potent immune activation contemporaneous to a dominant NK cell-dependent antiangiogenic effect in the B16 murine model of melanoma. Consequently, FIST prevent tumor formation not only in immunocompetent mice but also in several immunodeficient mice, whereas mice with NK defective functions such as nonobese diabetic–severe combined immunodeficient (NOD-SCID) mice and Rag2/c KO mice developed tumors. In the chapter 5, I generate and characterize FIST-stimulated B cells. FIST-stimulated B cells upregulate co-stimulatory molecules, activation markers and MHC class II molecule expression, which is also supported by robust hyperactivation of Jak/STAT signaling pathway. FIST-stimulated B cells act as effective APC that induce the activation and cell proliferation of antigen-specific CD4+ and CD8+ T cells. Interestingly, FIST-stimulated B cells confer complete protective immunity to EG.7 tumor challenge in vivo. Therefore, FIST can also be used as stimulator to generate B cells with APC features useful for the cell-based therapy of cancer. In conclusion, these bi-functional chimeric proteins are potential biopharmaceuticals for the therapy of cancer.
La 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 (TGFRII 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.

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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.

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In this thesis we show the generation and characteriation of specific GPI-anchored cell surface markers. The creation of these constructs has also allowed the quantification of levels of GPI linked protein shedding into the cell-culture media. Experiments presented in the thesis also show that the addition of a GPI anchor to Growth hormone (GH) appears to reduce expression in the media and that these molecules retain the GPI moiety. We speculated that anchoring cytokine ligands to the cell surface might also modulate receptor mediated signalling. To test the effect of GPI modification on cytokine ligands we fused the GH, and Ob mRNA sequences to the GPI anchor signal sequence of the naturally GPI-anchored protein, Thy-l. To our surprise we found that endogenously expressed GPI anchored cytokines acted as potent non-competitive cytokine receptor antagonists. Furthermore, we demonstrate that GPI anchored GH from a stable CHO cell-line can be purified and reinserted into mammalian cell membranes where they are able to induce receptor mediated signalling. Mammalian cell expression technologies are reliant on methods for obtaining high-yielding stable clones. This thesis describes the development of a high-throughput screening procedure based on the direct pull-down and enrichment of stably transfected cells using magnetic activared cell sorting (MACS). Results presented in this thesis demonstrate that it may be possible to use this procedure to isolate transfected cells from a heterogeneous population and to preferentially recover those rare cell populations which express higher levels of recombinant biopharmaceutical. Thus, GPI anchored markers may be used to facilitating facile cloning and identify population heterogeniety.

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19

Byun, 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.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2010.
"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.

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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.

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Rodrigues, 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/.

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A L-Asparaginase II de Escherichia coli (EcA II) é uma enzima amplamente utilizada no tratamento da Leucemia Linfoblástica Aguda (LLA), atuando na depleção do aminoácido L-asparagina, o qual é fundamental para a multiplicação das células cancerosas. Contudo, o tratamento com a EcA II está associado a altos índices de hipersensibilidade, devido à formação de anticorpos anti-L-asparaginase e à clivagem da enzima pelas proteases sanguíneas asparagina endopeptidase (AEP) e catepsina B (CTSB). Também ocorre neurotoxicidade associada ao efeito L-glutaminase da enzima. O principal objetivo do presente trabalho é a obtenção de mutantes da EcA II (gene ansB) com equivalente eficiência catalítica, maior resistência à clivagem proteolítica e menor atividade glutaminase. Para este propósito, através da reação em cadeia da polimerase propensa a erro (epPCR) do gene ansB, foi construída uma biblioteca de 1128 clones expressos no vetor pET15b em BL21(DE3). Nenhum mutante com atividade asparaginásica equivalente à EcA II selvagem apresentou atividade glutaminásica inferior à esta. Dentre os clones triados obtivemos um mutante (T161I) resistente à clivagem proteolítica pela CTSB e dois mutantes (Q190L e P40S/S206C) resistentes à clivagem proteolítica por ambas AEP e CTSB. Estes três mutantes apresentaram atividade asparaginásica e glutaminásica equivalentes a EcA II selvagem. Nossos resultados mostram promissoras possibilidades de EcA II mutantes com maior estabilidade frente às proteases sanguíneas humanas e possivelmente menos imunogênicas.
Escherichia 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.

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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.

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Les récents progrès instrumentaux en spectrométrie de masse, notamment en terme de- rapidité de balayage et de résolution, ont permis l'émergence de l'approche « data independent acquisition» (DIA). Cette approche promet de combiner les points forts des approches « shotgun » et ciblées,mais aujourd'hui l'analyse des données DIA reste compliquée. L'objectif de cette thèse a été de développer des méthodes innovantes de spectrométrie de masse, et en particulier d'améliorer l'analyse des données DIA. De plus, nous avons développé une approche originale Top 3-ID-DIA, permettant à la fois un profilage complet des protéines de la cellule hôte (HCP) ainsi qu'une quantification absolue d'HCP clés dans les échantillons d'anticorps monoclonaux (mAb), au sein d'une même analyse.Cette méthode est prête à être implémentée en industrie, et pourrait fournir un support en temps réel aux développements du procédé de production de mAb, ainsi que pour évaluer la pureté des biomédicaments
Recent 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

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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.

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Affibody® molecules are small therapeutic proteins which mimics antibody functionality. This is a report of several methods for increasing productivity and yield in recombinant production of Affibody® molecules. This literature study shows several steps in the production line which can be optimised, several novel methods for cultivating and harvesting cells and purication of proteins. There is also a section about validation of therapeutic protein production according to The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) are presented.

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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.

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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.

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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.

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El present treball es centra en l’estudi de la producció de proteïnes recombinants en línies cel·∙lulars de mamífer. Concretament, s’ha realitzat l’estudi de tres estratègies de bioprocés, totes elles basades en el cultiu de cèl·∙lules HEK293. Com a proteïna model per a l’expressió de proteïnes heteròlogues s’ha triat la proteïna CapPCV2, la qual conforma la càpsida viral del Circovirus porcí serotip 2 (PCV2). Aquest virus és l’agent causal de PCVDS (porcine circovirus diseases o malaties derivades de circovirus porcí). Aquest terme engloba un conjunt de malalties i síndromes que tenen un elevat impacte econòmic en la indústria porcina. El projecte s’ha enfocat des de la perspectiva de desenvolupament i optimització del bioprocés i, en conseqüència, l’increment de la producció volumètrica ha estat la força impulsora de tot el treball. En primer lloc es presenten els estudis per a la selecció del medi de cultiu i suplements nutricionals. El creixement cel·∙lular depèn en gran mesura de les característiques nutricionals i fisicoquímiques del medi en que se les cultiva. Per tant, trobar el medi adequat és un dels factors clau per a l’expansió del cultiu cel·∙lular. L’estudi inicial de medis de cultiu va permetre augmentar sis vegades la densitat de cèl·∙lules viables en comparació al medi original en que es cultivaven. D’altra banda, s’han explorat diferents estratègies de cultiu, i com a resultat s’ha implementat una estratègia de fed-‐batch que ha permès arribar a densitats cel·∙lulars de 26.8x106 cell/mL. En el segon i tercer capítol de resultats, s’avaluen tres estratègies diferents per a la producció de la proteïna recombinant CapPCV2 (r-‐CapPCV2). La primera estratègia ha estat la infecció de cèl·∙lules HEK293 amb un vector adenoviral que codifica el gen de la CapPCV2 (vector generat dins del treball d’aquesta tesis doctoral). Els paràmetres d’infecció s’han estudiat en profunditat per tal de trobar els paràmetres d’infecció (medi de cultiu, MOI (multiplicitat d’infecció), TOI (temps d’infecció) i TOH (temps de recollida)) per a la millora de la producció de la proteïna i el vector adenoviral. La segona i tercera estratègia estan basades en la generació de línies cel·∙lulars estables. Concretament, s’ha generat una línia cel·∙lular productora de r-‐CapPCV2 a partir de la integració a l’atzar del vector plasmídic en el genoma de la cèl·∙lula. D’altra banda, s’han generat línies cel·∙lulars amb la integració dirigida del gen en llocs prèviament caracteritzats com d’altra transcripció genètica. La integració dirigida s’ha efectuat mitjançant la tecnologia RMCE (recombinant mediated cassette exchange, o bescanvi de casset mitjançada per recombinació). Després de la comparació de les productivitats específiques i volumètriques aconseguides amb cada estratègia, el millor productor va ser seleccionat. Nogensmenys, r-‐CapPCV2 es produeix en quantitats molt baixes i per tant no ha sigut possible dissenyar un procés de producció rentable i altres alternatives de producció s’haurien d’estudiar en un futur. Finalment, l’estudi d’un comportament metabòlic particular observat en les cèl.lules en cultiu s’ha adreçat des d’una perspectiva fisiològica i metabòlica. A certes condicions extracel·∙lulars, s’ha observat que les cèl·∙lules HEK293 poden consumir de manera simultània glucosa i lactat durant el seu creixement exponencial. Després d’un ampli estudi d’aquestes condicions, s’ha determinat que el canvi de la producció d’àcid làctic (que és el principal problema dels cultius d’alta densitat de cèl·∙lules de mamífer) cap al consum d’aquest metabòlit pot ser generat des de el començament del cultiu quan el pH és de 6.6 i la concentració de lactat és de 4-‐8mM. En aquestes condicions, ni el creixement cel·∙lular ni la producció de proteïna es veuen afectades negativament. A la llum d’aquests resultats, es genera la hipòtesi de que les cèl·∙lules HEK293 poden co-‐transportar el lactat extracel.lular i els protons com un mecanisme de detoxificació del pH. D’altra banda, l’aplicació de l’anàlisi de balanç de fluxos (FBA) ha revelat que quan la glucosa i el lactat es consumeixen simultàniament s’aconsegueix un metabolisme “equilibrat”, és a dir els fluxos de la glicòlisi i el cicle TCA esdevenen similars, evitant l’acumulació de piruvat en el citosol, la seva transformació a làctic i finalment la secreció d’aquest metabòlit. Aquest comportament és totalment oposat al que s’observa de forma general en els cultius de cèl.lules de mamífer en creixement exponencial, on els elevats fluxos de la glicòlisi troben una limitació en els fluxos d’entrada a la mitocòndria (és a dir, del cicle TCA) i conseqüentment el lactat és produït i secretat al medi. La construcció d’un model metabòlic i l’aplicació de FBA permetrà fer prediccions in silico de comportaments metabòlics causats per la sobreexpressió o el silenciament de gens diana. Aquesta estratègia obre la possibilitat de generar línies cel·∙lulars que presentin un metabolisme optimitzat per tal d’estudiar estratègies de cultiu més eficients per a l’increment de la densitat cel·∙lular i productivitat de proteïna recombinant.
The 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.

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26

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/.

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A L-Asparaginase (L-ASNase) é uma enzima tetramérica bacteriana, utilizada em sessões de quimioterapia. Essa enzima depleta os aminoácidos asparagina (Asn) e glutamina (Gln), transformando-os em aspartato (Asp) ou glutamato (Glu), respectivamente, e em amônia. Contudo, a L-ASNase pode induzir resposta imune, levando à produção de anticorpos antiasparaginase, uma causa importante de resistência ao medicamento. Uma L-ASNase ideal seria aquela com alta atividade e estabilidade e baixo potencial imunogênico, porém, as L-ASNases utilizadas na terapêutica não reúnem essas características simultaneamente. Por essa razão, o presente trabalho utilizou técnicas de mutagênese randômica, a fim de criar uma nova proteoforma de L-ASNase de E. chrysanthemi com uma melhor atividade e estabilidade. Além disso, foram estudadas condições de cultivo em agitador metabólico, visando à otimização de condições de produção. Foi criada uma biblioteca com 1.056 clones, e desses, 19 foram selecionados por apresentarem atividade superior ou igual à enzima selvagem quando dosada em extrato bruto. Dentre eles, dois mutantes se destacaram por apresentarem a atividade específica glutaminásica diferente da enzima selvagem. Análises in silico indicam que o mutante 9-6D apresentou diminuição de desordem estrutural e epítopos imunogênicos. O mutante 9-5F demonstrou uma diminuição da porcentagem da atividade glutaminásica quando comparada a enzima selvagem. O estudo de produção do mutante 9-5F indicou que a temperatura de indução, seguida da concentração do indutor, são os parâmetros mais relevantes para a otimização da produção de L-ASNase de E. chrysanthemi mutante.
L-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.

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27

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.

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A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

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28

Pow, 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/.

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A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the â-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) Escherichia coli proteins were used as model interaction partners for developing the system. These proteins drove effective â-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other â-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent â-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wildtype Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

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29

Petri, 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.

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The 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.

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30

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/.

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Cell line engineering strategies for improved biopharmaceutical production in mammalian cells often involve the expression of one/multiple genes to try and improve the cellular processes involved in recombinant protein production. Most strategies are relatively simple, involving the use of a strong constitutive promoter for expression of one or more proteins to help increase production. Results often vary and can be cell line and product specific and mean a generic strategy is unlikely to be found. There is a need for more sophisticated expression systems which can express multiple genes but in a controlled fashion and tuned to meet the needs of a specific product. This thesis can be split into two distinct parts but both concern the expression of multiple genes in mammalian cells and recombinant protein production. A tunable mammalian expression system for multi-gene engineering composed of elements of the mammalian unfolded protein response has been developed. ATF6 (activating transcription factor 6) and its binding element (ERSE – ER stress response element) were used to control the expression of the reporter proteins SEAP (secreted alkaline phosphatase) and GFP (green fluorescent protein). By expressing different amounts of ATF6 and by inserting different numbers of ERSEs upstream of a SV40 (Simian virus 40) promoter, driving SEAP/GFP gene transcription, the level of reporter protein expression could be manipulated in a controlled fashion. The system was capable of controlled/tunable expression of both reporter proteins when expressed alone and when they were co-expressed. This a novel use for ATF6 and ERSE and the first step towards the development of a tunable mammalian expression system for multi-gene engineering. This system could also be easily modified to include or use different transcription factors and binding sites as well as having the potential to use completely synthetic components. This work also showed that the presence of ‘promoter interference’ (the negative influence of one promoter on another) could be used to our advantage to increase the range of expression. The SV40 early, human CMV (cytomegalovirus) major immediate-early and human EF1α (elongation factor 1 alpha) are constitutive promoters frequently used in recombinant protein production. The former being used mainly for expression of selection genes and the latter two for strong expression of recombinant proteins. The differences in the strengths of the promoters was demonstrated in CHO (Chinese hamster ovary) cells (CMV > EF1α > SV40) and also their abilities to negatively affect the expression from a co-expressed promoter. The negative influence of one promoter on another is termed ‘promoter interference’. The CMV promoter was shown to have the greatest negative effect on expression from another promoter, decreasing both SEAP mRNA and protein expression, while the SV40 had the least. SEAP expression from the SV40 was reduced the most by the presence of a competing promoter. The level of interference inflicted by a competing promoter (CMV > EF1α > SV40) seemed to be relative to its strength. This is the first time these three important promoters have been compared in a way which not only demonstrates their relative strengths but also their ability to interfere with another promoter when present in the same transient expression system. This also has implications for their use in multi-gene engineering strategies if there is a need for controlled/tunable expression of multiple genes. The work with ATF6 and ERSE showed how ‘promoter interference’ could be used to our advantage and not necessarily be just a negative occurrence. One hypothesis for why promoter interference occurs is there is competition between promoters for shared transcription factors (TFs). The promoters were analysed for potential transcription factor binding sites (TFBSs) using the programs MatInspector and ModelInspector. The analysis showed that the SV40 promoter had the least number and variety of potential TFBSs. Both the CMV and EF1α had greater numbers and variety of potential TFBSs. All three promoters had common potential TFBSs but the SV40 promoter shared a greater proportion of its sites with the other two promoters. The number of potential TFBSs and the proportion which were shared reflected both the strength and the ability of a promoter to interfere with another. All three contained TFBSs for the SP1 (specificity protein 1) family of TFs and over-expression of SP1 counter-acted the effects of promoter interference showing that it can affect the expression of all three promoters. However, promoter interference will involve more than just a single TF and also more than just competition for transcriptional activators. This is the first time these three promoters have been compared in terms of the potential TFBSs they contain. The TFBS analysis highlighted the complexity in the control of these promoters and with the effects of promoter interference means that they will be ill suited for the controlled expression of multiple genes without modification. The work in this thesis was directed towards the controlled expression of multiple genes in mammalian cells for recombinant protein production. We have presented one novel way of controlling the expression of one/two genes with the rest of the thesis looking at the phenomenon of ‘promoter interference’ between three commonly used promoters. This thesis tries to highlight the importance of multi-gene expression systems as well showing that these three promoters may not be suitable without further modification and also the importance of considering promoter interactions when more than one is present in the same system. The switch to completely synthetic multi-gene control systems is something we envisage happening in the future as the complexities and capabilities of these systems grow.

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31

Kohli, 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.

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One of the pathological hallmarks of Alzheimer's disease (AD) is the amyloid plaque deposition in aging brains by aggregation of amyloid-? (A?) peptides. In this study, the effect of chloroplast derived myelin basic protein (MBP) fused with cholera toxin subunit B (CTB) was investigated in advanced diseased stage of human and mouse AD brains. The CTB-fusion protein in chloroplasts facilitates transmucosal delivery in the gut by the natural binding ability of CTB pentameric form with GM1 receptors on the intestinal epithelium. Further, bioencapsulation of the MBP within plant cells confers protection from enzymes and acids in the digestive system. Here, 12-14 months old triple transgenic AD mice were fed with CTB-MBP bioencapsulated in the plant cells for 3 months. A reduction of 67.3% and 33.3% amyloid levels in hippocampal and cortical regions, respectively were observed by immunostaining of brain sections with anti- A? antibody. Similarly, 70% decrease in plaque number and 40% reduction of plaque intensity was observed through thioflavin S (ThS) staining that specifically stains amyloid in the AD brain. Furthermore, ex vivo 3xTg AD mice brain sections showed up to 45% reduction of ThS stained amyloid levels when incubated with enriched CTB-MBP in a concentration dependent manner. Similarly, incubation of enriched CTB-MBP with ex vivo postmortem human brain tissue sections with advanced stage of AD resulted up to 47% decrease of ThS stained amyloid plaque intensity. Lastly, lyophilization of plant material facilitates dehydration and long term storage of capsules at room temperature, in addition to increasing CTB-MBP concentration by 17 fold. These observations offer a low cost solution for treatment of even advanced stages of the AD by facilitating delivery of therapeutic proteins to central nervous system to address other neurodegenerative disease.
M.S.
Masters
Molecular Biology and Microbiology
Medicine
Biotechnology

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32

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.

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Control and analysis of protein aggregation is an increasing challenge to biopharmaceutical research and development. Therefore it is important to understand the interactions, causes and analysis of particles in order to control protein aggregation to enable successful biopharmaceutical formulations. This work investigates the role of different non-ionic surfactants on protein conformational stability, as assessed by HSDSC, and on protein size stability as assessed by Dynamic Light Scattering (DLS), HIAC and MFI. BSA and IgG2 were used as model proteins. Thermal unfolding experiments indicated a very weak surfactant-immunoglobulin IgG2 interaction, compared to much stronger interactions for the BSA surfactant systems. The DLS results showed that BSA and IgG2 with different surfactants and concentration produced different levels of particle size growth. The heat treatment and aging of samples in the presence of Tween 20, Tween 80, Brij 35 and Pluronic F-68 surfactants led to an increase in the populations of larger particles for BSA samples, whereas IgG2 systems did not notably aggregate under storage conditions MFI was shown to be more sensitive than HIAC technique for measuring sub-visible particles in protein surfactant systems. Heat treatment and storage stress showed a significant effect on BSA and IgG2 protein sub-visible particle size stability. This work has demonstrated that both proteins with different Tween 20, Tween 80, Brij 35 and Pluronic F-68 concentrations, have different level of conformational and size stability. Also aging samples and heating stress bears the potential to generate particles, but this depends on surfactant type. Poor predictive correlations between the analytical methods were determined.

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33

Silva, Bruna Daniela Gonçalves da. "Nanopartículas lipídicas para a administração de produtos biofarmacêuticos." Master's thesis, [s.n.], 2015. http://hdl.handle.net/10284/5187.

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Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
Os 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.

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34

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/.

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O fator VIII de coagulação (FVIII), recombinante ou purificado de plasma, é o biofármaco necessário para o tratamento da hemofília A, a doença hemorrágica mais freqüente em humanos. O método tradicional para a purificação de FVIII parte de crioprecipitado de plasma e precipitação alcoólica. No Instituto Butantan, foi proposto um método alternativo, utilizando somente cromatografia para esta purificação. Este projeto teve por objetivo comparar dois métodos cromatográficos de purificação do FVIII: 1 - gel filtração direta do plasma e 2 - pré-purificação de FVIII do plasma por cromatografia de troca aniônica, seguida de gel filtração. A purificação foi analisada por dosagens de atividade específica de FVIII e presença de outras proteínas da cascata de coagulação nas frações de cromatografia. Foram realizadas clonagem de fragmentos gênicos de FVIII e expressão de fragmentos protéicos para imunização de animais. Os soros com anticorpos policlonais anti-FVIII foram usados em ensaios de \"western blot\" para detectar as cadeias de FVIII ou degradação.
Coagulation 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.

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35

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.

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This dissertation has focused on studying the effect of four glycine derivatives on the solid state properties of mannitol, glycine, and sucrose when freeze dried into blended mixtures. The primary goal was to assess their value for use in the stabilisation of vaccines in the solid state, by examining key physical and chemical characteristics, which have been documented to be beneficial to the stabilisation of biopharmaceutical formulations. The novel excipients; dimethyl glycine, and trimethyl glycine, were shown to retard the crystallisation and increase the overall glass transition temperature, of mannitol, when freeze dried as evidenced by DSC and Powder X-ray diffraction. Mannitol’s glass transition temperature increased from 100C to 12.650C and 13.610C when mixed with methyl-glycine and dimethyl glycine respectively. The glycine derivatives did not show the same effect on sucrose which remained amorphous regardless of the concentration of the other excipient. The different behaviour with the sucrose system was thought to be due to relatively high glass transition temperature of sucrose. Conversely glycine remained highly crystalline due it’s relatively low glass transition temperature. The novel excipient formulations were also assessed for their effect on the aggregation of the adjuvant aluminium hydroxide when freeze dried by Dynamic Light Scattering (DLS).The formulations containing the glycine derivatives all caused a decrease in the aggregation size of the adjuvant from ~26 μm, to 185 nm in the presence of methyl glycine. The effects of lysozyme and viral antigen on the adjuvants were also examined showing that the addition of the virus did not affect the size of the aggregates formed, however lysozyme showed significant decreases in the aggregates formed. Examination of the freezing method were also made showing that faster freezing rates produced smaller aggregates of the adjuvant. When investigating the rate at which the excipients lost water during secondary drying there was evidence of the formation of hydrates of glycine, trimethyl glycine, and mannitol has shown that the glycine derivatives have attributes which would be beneficial in stabilising vaccines in the solid state when freeze dried.

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36

Sunley, 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.

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Mild hypothermic conditions (30ºC to 33ºC) have previously been shown to increase cell specific productivity (Qp) of recombinant proteins from mammalian cells. However, this is often associated with a lower growth rate which off-sets any potential advantage of higher product titres. This thesis describes the isolation of a novel population of Chinese Hamster Ovary (CHO) cells that have been adapted to low temperature growth by continuous subculture at low temperature for a duration of 400 days. This adapted cell population achieved a growth rate 2-fold greater than non-adapted cells under low temperature conditions (32ºC) while maintaining an elevated level of cell specific expression of recombinant beta-interferon. The volumetric titre of beta-interferon was enhanced by 70% in stationary cultures and by more than 2-fold by application of a temperature-shift strategy involving a growth to production phase. However, the low temperature-adapted cells were fragile and demonstrated an increased sensitivity to hydrodynamic stress in agitated cultures. This problem, caused by a weakened vimentin intermediate filament network, was resolved by the use of macroporous microcarriers which were demonstrated to entrap and protect the cold-adapted cells. Cold-adapted microcarrier cultures were able to achieve high cell densities (greater than 5x10^6 nuclei/mL) cultures under hypothermic conditions. This resulted in a 3-fold enhancement of volumetric titre of monomeric beta-interferon compared to the original control culture at 37ºC.

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37

Charlton, Adam. "Improved production of biopharmaceuticals by site-specific cleavage of fusion proteins expressed in Escherichia coli." 2008. http://hdl.handle.net/2440/59637.

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Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library.
The 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.
http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1325381
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

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38

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.

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Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library.
The 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

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39

"Optimization of a Viral System to Produce Vaccines and other Biopharmaceuticals in Plants." Doctoral diss., 2017. http://hdl.handle.net/2286/R.I.46337.

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abstract: Plants are a promising upcoming platform for production of vaccine components and other desirable pharmaceutical proteins that can only, at present, be made in living systems. The unique soil microbe Agrobacterium tumefaciens can transfer DNA to plants very efficiently, essentially turning plants into factories capable of producing virtually any gene. While genetically modified bacteria have historically been used for producing useful biopharmaceuticals like human insulin, plants can assemble much more complicated proteins, like human antibodies, that bacterial systems cannot. As plants do not harbor human pathogens, they are also safer alternatives than animal cell cultures. Additionally, plants can be grown very cheaply, in massive quantities. In my research, I have studied the genetic mechanisms that underlie gene expression, in order to improve plant-based biopharmaceutical production. To do this, inspiration was drawn from naturally-occurring gene regulatory mechanisms, especially those from plant viruses, which have evolved mechanisms to co-opt the plant cellular machinery to produce high levels of viral proteins. By testing, modifying, and combining genetic elements from diverse sources, an optimized expression system has been developed that allows very rapid production of vaccine components, monoclonal antibodies, and other biopharmaceuticals. To improve target gene expression while maintaining the health and function of the plants, I identified, studied, and modified 5’ untranslated regions, combined gene terminators, and a nuclear matrix attachment region. The replication mechanisms of a plant geminivirus were also studied, which lead to additional strategies to produce more toxic biopharmaceutical proteins. Finally, the mechanisms employed by a geminivirus to spread between cells were investigated. It was demonstrated that these movement mechanisms can be functionally transplanted into a separate genus of geminivirus, allowing modified virus-based gene expression vectors to be spread between neighboring plant cells. Additionally, my work helps shed light on the basic genetic mechanisms employed by all living organisms to control gene expression.
Dissertation/Thesis
Doctoral Dissertation Microbiology 2017

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Das, Anindita. "Nanoparticle Mediated Suppression of Protein Aggregation." Thesis, 2015. http://etd.iisc.ernet.in/2005/3521.

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The increasing demands for biopharmaceuticals to treat different diseases have raised concerns about controlling the quality and efficacy of such pharmaceuticals. The design and formulation of a stable protein or peptide based biopharmaceutical runs into the limitation that at high concentrations (> 100 mg/ml) or during long storage process the drug undergoes aggregation. During synthesis, purification, storage or packaging of these drugs different kinds of stresses like chemical, oxidative, thermal, shear, etc. are encountered. These stresses promote the non-native aggregation of protein and peptide based drugs. Injection or administration of such drugs if contaminated with aggregates causes patient discomfort or development of an antibody which can adversely affect patient’s conditions. This brings out the necessity of finding a way so that such aggregation is avoided. Nanoparticles have been used as vehicles for drug delivery and diagnostic agents in biology for a while. The surface of the nanoparticles is known to adsorb small as well as large molecules with different kinetics and energetics of interaction. I have used nanoparticles to adsorb proteins to protect them against aggregation when they are subjected to denaturing conditions. The effectiveness of the nanoparticles in stopping protein aggregation, recovery of the proteins and reversibility of the adsorption process, the catalytic activity of the proteins before and after adsorption on the surface have all been studied in details. The work described here has been divided in 8 chapters and the contents of each chapter are described below. In Chapter 1 I have provided a brief introduction to the protein aggregation problem. The motivation and scope of the current work has been presented in this chapter. Materials and methods have been described in Chapter 2. Synthesis of gold and silica nanoparticles, their characterization and stability under experimental conditions have been illustrated in this chapter. The spectroscopic assays and techniques which I have used to study the effect of gold and silica nanoparticles on protein aggregation have been discussed at lengths in this chapter. In Chapter 3 I have demonstrated the effect of gold nanoparticles on thermal aggregation of alcohol dehydrogenase (ADH). The size of the nanoparticle was varied in the range of 15-60 nm and the effect was measured by various spectroscopic assays and techniques. I have observed that gold nanoparticles prevent thermal aggregation of ADH and the efficiency is high. Gold nanoparticles in nanomolar or even picomolar concentrations are capable of preventing the aggregation of ADH at micromolar concentrations. In Chapter 4 the role of gold nanoparticles as suppressor of protein aggregation was extended to another protein, insulin. Chemically induced aggregation of insulin using dithiothreitol (DTT) in the presence of gold nanoparticles was studied in the same manner as was done for ADH. Similar prevention property of gold nanoparticles was established by making the observation independent of the method of denaturation or the type of protein used in the prevention experiments. In Chapter 5 huge second harmonic light scattering (SHS) signal from pure gold nanoparticles has been used to measure the free energy of interaction of ADH and insulin with nanoparticles in solution, for the first time. The change in the second harmonic scattered signal was monitored which decreased steadily as a function of added protein concentration to the aqueous solution of gold nanoparticles. The fitting of the second harmonic signal decay was done with a modified Langmuir adsorption isotherm to extract the free energy change in the interaction and the number of protein molecules adsorbed on the surface. In Chapter 6 I have demonstrated a way to recover the adsorbed ADH and insulin from the gold nanoparticle surface and tested the activity of ADH by an assay. The structure of the proteins in the adsorbed state has been probed by CD spectroscopy and described in this chapter. It is found that ADH retains its activity in the adsorbed state. Both the proteins retain the native secondary structures in their adsorbed state. However, the structures change drastically under denaturing conditions. In Chapter 7 the effect silica nanoparticles which are known to have hydrophilic surface has been examined on the aggregation of ADH and insulin in pretty much the same way as was done with gold nanoparticles. The efficiency of silica nanoparticle was found to be lower compared to gold nanoparticles. In addition, the size dependency of prevention efficiency of silica and gold nanoparticles was found to be completely opposite to each other. In Chapter 8 I have presented the overall summary and possible future directions of this work

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Dissertations / Theses on the topic 'Biopharmaceutical proteins'

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