Дисертації: "Chromatographic purification"

1

Hamdi, Anis. "Novel Chromatographic methodology for virus particles purification." Master's thesis, Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica António Xavier, 2016. http://hdl.handle.net/10362/64186.

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"Virus based biopharmaceuticals are considered the most dynamic adopted tools in modern therapeutic medicine. Their use is increasing in the fields of vaccination and gene therapy. Membrane chromatography is becoming an attractive alternative t ool that can be used for virus purification due to its scalability, potential for optimization and economical features, allowing higher productivities with lower DSP costs. (…)"
N/A

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2

Mekaoui, Nazim. "Contribution à l'étude de la chromatographie à contre-courant : partage de composés ionisables, nouvelles colonnes et purification séquentielles." Thesis, Lyon 1, 2012. http://www.theses.fr/2012LYO10249/document.

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La chromatographie à contre courant (CCC) est une technique de purification chimique préparative quitravaille avec un système biphasique liquide. Une phase est la phase mobile, l'autre phase est la phasestationnaire. Il n'y a aucun support solide: un champ de force centrifuge est utilisé pour maintenir en place laphase stationnaire. Ce travail est une contribution à l'étude de la purification préparative par CCC. Après uneimportante étude bibliographique des procédés de purification en continu tant en CCC qu'autres, il est montréque la méthode dite "multi-dual-mode", ou MDM, est une solution possible. Elle consiste à utiliser le fait queles deux phases liquides peuvent servir de phase stationnaire: il suffit d'inverser le sens de circulation et lanature de la phase mobile (méthode dual-mode). Le mélange est séparé de façon classique pendant untemps chronométré, puis on inverse le rôle des phases: la phase mobile devient stationnaire et vice versa eton inverse également le sens de circulation (ascendant devient descendant ou vice versa). On sort lescomposants du mélange soit d'un coté de la colonne CCC, soit de l'autre. La méthode est mise en oeuvrepour purifier le Bleu de Coomassie en le débarassant des ses composés polaires (d'un coté) et apolaire (del'autre coté de la colonne et en accumulant dans la colonne la fraction de polarité intermédiaire, fractiond'intérêt. Une nouvelle colonne hydrostatique de petit volume (30 mL) a également été testée: elle permetde tester un nouveau système liquide très rapidement
Counter-current chromatography (CCC) is a preparative purification technique that works with the twoliquid phases of a biphasic liquid system. One phase is used as the mobile phase when the other phase isused as the stationary phase. There is no solid support: centrifugal fields are used to obtain a support-freeliquid stationary phase. This work contains an exhaustive bibliographic study of what can be found in theliterature concerning continuous chromatographic processes. The multi-dual-mode (MDM) process was foundto be the best one able to purify large amount of crude mixtures. The MDM method starts with a classicalseparation of the mixture followed by a switch of both the liquid phase nature and the flowing direction. Themobile phase flowing e.g. in a descending direction becomes the stationary phase. The previous stationaryphase becomes the mobile phase flowing in the ascending direction (or vice versa). The purified compoundsof the introduced mixture are eluted at one side of the column or the other according to their polarity. TheMDM method was used to purify a crude sample of Coomassie Blue: the polar part of the dye was eluted atthe column top (or head) and the apolar part at the column bottom (or tail) while the essential part of the dyewas trapped inside the CCC column. The work also presents a new small volume (30 mL) hydrostatic CCCcolumn. It is shown that this column could be used to test quickly the potential of a given biphasic liquidsystem

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3

Balci, Oguz. "Affinity chromatographic purification of recombinant human growth hormone." Master's thesis, METU, 2008. http://etd.lib.metu.edu.tr/upload/2/12609269/index.pdf.

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The purpose of the study is to purify human growth hormone from the fermentation broth by affinity chromatography. For this purpose, human growth hormone specific oligonucleotide aptamers are selected among an aptamer library
selected oligonucleotides were synthesized and used as ligands. Effect of pH on ligand-human growth hormone complex formation was investigated and the highest complex formation was obtained at pH= 7.0. Human growth hormone is separated from the fermentation broth with 99.8% purity and 41% overall yield. The equilibrium data obtained was described by Langmuir type isotherm where saturation constant (q0) and affinity constant (K) are calculated as 0.338 mg hGH/µ
mol aptamer and 0.059 mg hGH/ml, respectively. Further, equilibrium data obtained using aptamer affinity column was described by Langmuir type isotherm where saturation constant (q0) and affinity constant (K) are 0.027 mg hGH/µ
mol aptamer and 1.543 mg hGH/ml, respectively. It is possible that, selected aptamer can be used for purification of bulk amounts of recombinant human growth hormone by using aptamer affinity chromatography.

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4

Ramat, Fabien M. "Protein purification using expanded bed chromatography." Link to electronic thesis, 2003. http://www.wpi.edu/Pubs/ETD/Available/etd-0114104-114704.

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Thesis (M.S.)--Worcester Polytechnic Institute.
Keywords: zirconia; protein purification; anion exchange; chicken egg white; expanded bed chromatography. Includes bibliographical references (p. 83-86).

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5

Nakamura, Koji. "Studies on High-performance Affinity Chromatography : Preparation of the Chromatographic Gels, Evaluation of the Chromatographic Conditions, and the Application to Purification of Enzymes." Kyoto University, 2004. http://hdl.handle.net/2433/148344.

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6

Bergs, Dominik [Verfasser]. "A contribution to chromatographic purification of natural products / Dominik Bergs." München : Verlag Dr. Hut, 2013. http://d-nb.info/1045989150/34.

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7

Ramat, Fabien M. "Protein purification using expanded bed chromatography." Digital WPI, 2004. https://digitalcommons.wpi.edu/etd-theses/92.

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Expanded bed chromatography using ion-exchange media is a powerful first step in purification processes. Expanded bed chromatography can be used to extract components from complex and viscous solution. This can be achieved because of the void created between adsorbent particles where as in packed bed chromatography, the adsorbent is too compact and dense for a complex feed stock to flow through. Expanded bed chromatography was used to purify bovine serum albumin (BSA) from chicken egg white (CEW). The high viscosity of CEW presents a unique challenge for efficient large-scale protein purification. This project aimed to optimize and evaluate a separation method that is believed to be particularly suitable for high viscosity solutions: expanded-bed ion exchange chromatography. The BSA was admixed into the CEW and the solution was pumped through the column for purification. The media used in the column was Streamline DEAE which is an anion-exchanger. The yield obtained was 85% and the purity was 57%. A mathematical model to understand and predict the behavior of expanded bed chromatography was developed to provide an estimation of the breakthrough curves obtained for BSA. A small sized porous dense adsorbent was also synthesized to enhance the purification process. This zirconia-based adsorbent allows use of higher flow velocities that is a key factor when working with viscous fluids such as chicken egg white.

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8

Pate, Martin Eric. "A practical investigation into the use of principal component analysis for the modelling and scale-up of high performance liquid chromatography." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322003.

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9

Kochendörfer, Kiara [Verfasser]. "Chromatographic Purification of an Intermediately Eluting Component from a Complex Mixture / Kiara Kochendörfer." München : Verlag Dr. Hut, 2017. http://d-nb.info/1126297976/34.

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10

Jin, J. "Lipid foulant interactions during the chromatographic purification of virus-like particles from Saccharomyces cerevisiae." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1302065/.

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The objective of this study was to understand the mechanism of lipid fouling in chromatography through the investigation of a hydrophobic interaction chromatography (HIC) operation. This was motivated by the need to understand this phenomenon during the manufacture of biological products such as vaccines. The systematic approach and novel analytical techniques employed create a unique platform to study fouling of other chromatographic adsorbents and process feed materials. HIC is employed as a primary capture step in the purification of yeast derived hepatitis R surface antigen (HBsAg), where the required cell disruption and detergent liberation steps release high levels of lipid content into the feed stream. From lipid- rich and lipid-depleted feedstocks, comparative analysis was able to quantify the deterioration in HIC performance (binding capacities, purities and recoveries) under successive cycles. Furthermore, a full mass balance on host lipids identified the highly hydrophobic triacylglyceride as the main foulant. Intra-particle distribution and progression of lipid fouling and its effects on material adsorption and diffusion were then examined under confocal laser scanning microscopy (CLSM). In addition, high- resolution scanning electron microscopy (SEM) images of the fouled bead (after 40 cycles) confirmed that a thick lipid layer was building up on the outer bead surface. Based on these findings, the mechanism of fouling was thought to be the rapid accumulation of lipid foulant at the rim of the bead, which was aggravated by the possible diffusion hindrance resulting from multi layer adsorption. Finally, pretreatments to reduce this mechanism of chromatography fouling were evaluated in terms of improvement on feed quality and HIC performance. Selective adsorbent polystyrene XAD-4 demonstrated promising lipid removal capabilities with satisfactory HBsAg VLP recoveries. The improved feed into the column resulted in a three-fold increase in product capacity, whilst the overall yield remained constant over 40 cycles.

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11

Maeda, Atsushi. "Production of 3-Ketocellobiose from Cellobiose Using Agrobacterium tumefaciens Cells and Its Chromatographic Purification." Kyoto University, 2003. http://hdl.handle.net/2433/149002.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第10278号
農博第1350号
新制||農||869(附属図書館)
学位論文||H15||N3799(農学部図書室)
UT51-2003-H699
京都大学大学院農学研究科応用生命科学専攻
(主査)教授松野隆一, 教授井上國世, 教授村田幸作
学位規則第4条第1項該当

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12

Ma, Chun-hang. "Partial purification and characterization of 1-aminocyclopropane-1-carboxylic acid n-malonyltransferase from etiolated mung bean." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B36181341.

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13

Bonturi, Nemailla 1985. "Purificação em etapa cromatográfica única de DNA plasmidial a partir do lisado neutralizado visando a sua aplicação em estudos de terapia e vacinação gênica." [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/266888.

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Orientadores: Everson Alves Miranda, Adriano Rodrigues Azzoni
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química
Made available in DSpace on 2018-08-18T15:34:36Z (GMT). No. of bitstreams: 1 Bonturi_Nemailla_M.pdf: 2076232 bytes, checksum: e3f4e56f400891ab77fe029e0c1f0899 (MD5) Previous issue date: 2011
Resumo: O número de estudos em terapia gênica com vetores plasmídiais (pDNA) têm aumentado nestes últimos anos. Como resultado, a demanda para preparações de pDNA em conformidade com as recomendações das agências reguladoras (EMEA, FDA) também aumentou. O DNA plasmidial é frequentemente obtido através da fermentação de Escherichia coli transformada e purificada por uma série de operações unitárias, incluindo a cromatografia. Este trabalho teve como objetivo o desenvolvimento de um processo cromatográfico para a recuperação e purificação do pDNA superenovelado (sc pDNA) a partir do lisado neutralizado. Os ligantes fenil (hidrofóbico) e mercaptopirimidina (tiofílico) foram imobilizados em matrizes de agarose e celulose. A seletividade destes ligantes para com o sc pDNA foi determinada através de estudos de adsorção utilizando citrato de sódio 1,5 mol/L e fosfato de potássio 2,0 mol/L como tampões de adsorção. A cromatografia com o adsorvente fenil-agarose e o citrato de sódio 1,5 mol/L permitiu recuperar 58% do pDNA sem contaminação por gDNA, proteínas e endotoxinas, sendo uma alternativa potencial para a recuperação primária do sc pDNA. O resultado mais promissor foi obtido com a cromatografia com o adsorvente mercaptopirimidina-agarose e fosfato de potássio 2,0 mol/L como tampão de adsorção. Este sistema tampão de adsorção/adsorvente permitiu a obtenção de pDNA com 100% de pureza e dentro das recomendações das agências reguladoras no tocante à contaminação por RNA e endotoxinas. Assim, este trabalho lançou as bases para o desenvolvimento de dois métodos cromatográficos para a recuperação primária ou purificação de pDNA diretamente do lisado neutralizado, ambos potencialmente aplicáveis em larga escala
Abstract: The number of studies in gene therapy with plasmid vectors (pDNA) has witnessed an increase in the recent years. As result the demand for preparations of pDNA in compliance with recommendations of the regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is oftenly obtained through fermentation of transformed Escherichia coli and purified by a series of unit operations, including chromatography. This work aimed the development of a chromatographic process for the recovery and purification of supercolied pDNA (sc pDNA) directly from neutralized cell lysate. Phenyl (hydrophobic) and mercaptopyrimidine (thiophilic) molecules immobilized in agarose and cellulose matrices were the ligands used to capture the pDNA. Their selectivity towards sc pDNA was evaluated through adsorption studies using sodium citrate 1.5 mol/L and potassium phosphate 2.0 mol/L as the adsorption buffers. The chromatography with the adsorbent phenyl-agarose and sodium citrate 1.5 mol/L was able to recover 58% of sc pDNA without gDNA, proteins and endotoxins contamination, being an potential alternative for the primary recovery of sc pDNA. The most promising result was obtained with the chromatography with mercaptopyrimidine-agarose and potassium phosphate 2.0 mol/L adsorpition buffer. With the latter buffer/adsorbent system it was possible to obtain in a single step pDNA with 100% purity and within the recommendations of regulatory agencies with regard to contamination by RNA and endotoxins. Thus, this work laid the basis for the development of two chromatographic process for the recovery or purification of pDNA directly from the neutralized lysate, both potentially applicable in larger scale
Mestrado
Desenvolvimento de Processos Biotecnologicos
Mestre em Engenharia Química

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14

Kouyoumdjian, Arthur Jean Michel. "The functionalisation and application of microporous micro-capillary films for the chromatographic purification of biomolecules." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/289020.

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Microporous walled micro-capillary films (MMCFs) are porous polymer films with embedded capillaries. MMCFs have been found to be suitable low-cost chromatography substrates capable of tolerating high flowrates, which suggested they might be a solution to the growing bottleneck in the downstream purification of biopharmaceuticals. However, MMCFs have been mainly tested for binary separations of model proteins and broader capabilities of this technology remain largely unknown. The experimental work presented in this thesis focused on developing MMCFs functionalised with different ligands and their subsequent testing as chromatography media for bioseparations. MMCFs were functionalised to form weak anion (MMCF-DEAE) and weak cation-exchangers (MMCF-gCM and MMCF-CA). Emphasis was placed on low-cost functionalisation methods amenable to single-use applications. To confirm the addition of functional groups on the membranes, a comprehensive characterisation routine was implemented. This included the use of spectroscopy, electron microscopy, elemental analysis and pH titration. The binding performance of these weak ion-exchangers was further assessed by static and dynamic adsorption of model proteins. Next, the functionalised MMCFs were tested for bioseparations with binary and complex mixtures, the latter being more relevant for industrial applications. It was found that MMCFs could selectively separate similarly charged biomolecules using optimised elution strategies. Further, it was observed that MMCF-gCM could capture lysozyme from chicken egg white at a near twenty-fold purity increase compared to the feed. Similarly, this weak cation-exchanger could recover antibodies from unfiltered mammalian cell lysate. The recovered biomolecules were then injected onto MMCF-DEAE to remove nucleic acid impurities in subtractive chromatography mode. Finally, MMCFs were explored for the first time as affinity chromatography supports. Bovine serum albumin (BSA) and Protein A were covalently coupled to the substrate and tested for the capture of relevant analytes. Indeed, it was found that MMCF-BSA could bind bilirubin and MMCF-ProtA could be used to recover antibodies. Given the high cost of Protein A, a cheaper synthetic ligand was coupled onto MMCFs and observed to successfully bind antibodies. Overall, this work has furthered the applications of MMCFs for bioseparations, demonstrating their great versatility and robustness. Furthermore, it opened the field for affinity-based MMCFs, which could have numerous applications in both research and industry. Extensive characterisation methods presented here will greatly simplify future studies with these membranes. While the binding capacity of the developed ion-exchangers was typically two orders of magnitude higher than non-porous MCFs (NMCFs), the low yield achieved with MMCFs currently precludes them from commercial applications. However, optimisation of MMCFs, as outlined in the future work, could make this support more commercially viable and leverage the numerous advantages offered by its unique geometry.

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15

Ma, Chun-hang, and 馬進恆. "Partial purification and characterization of 1-aminocyclopropane-1-carboxylic acid n-malonyltransferase from etiolated mung bean." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B36181341.

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16

Chapman, James M. "The preparation and evaluation of immobilized retinoid analogues in the affinity chromatographic purification of retinoid binding proteins /." The Ohio State University, 1990. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487678444256637.

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17

Kissounko, Natalia. "Study of dynamics in a reaction catalyzed by ht- and ps-ADH cloning, purification and preliminary x-ray screening /." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 80 p, 2008. http://proquest.umi.com/pqdweb?did=1605142871&sid=6&Fmt=2&clientId=8331&RQT=309&VName=PQD.

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18

Galiardi, Jackelyn. "Split Intein Applications for Downstream Purification and Protein Conjugation." The Ohio State University, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=osu1618931990774251.

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19

Kröner, Frieder [Verfasser], and J. [Akademischer Betreuer] Hubbuch. "Novel approaches in chromatographic purification process development for low concentrated proteins in complex mixtures / Frieder Kröner. Betreuer: J. Hubbuch." Karlsruhe : KIT-Bibliothek, 2013. http://d-nb.info/1046362674/34.

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20

Philander, Ghouwaa. "Development of a gas chromatographic technique for the analysis of some groundwater contaminants from fuel leaks and its application in a site-specific study." Thesis, University of the Western Cape, 2009. http://hdl.handle.net/11394/2613.

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Magister Scientiae - MSc
This study focuses on the development of a Direct Aqueous Injection Gas Chromatographic method with Flame Ionization Detection (DAI-GC/FID) for the analysis of MTBE and TBA. The analytical method was then applied in a site specific study where MTBE contamination was evident. The method achieved detection limits of 1 ppm for MTBE and 0.1 ppm for TBA. The method showed good precision, accuracy and selectivity. The method was selected primarily for its ability to simultaneously analyze MTBE and TBA. The result of the site specific study showed the persistence of high concentrations of MTBE and TBA at the source of contamination, whilst concentrations at the adjacent primary school dropped to below detection limits as a result of rapid natural attenuation. It was found that an overall decrease in MTBE concentrations was met with an increase in TBA concentrations; which is a direct indication of MTBE degradation. Despite the fact that problematic MTBE concentrations persist at the source of contamination, limited evidence of the persistence of MTBE contamination was identified at the adjacent primary school. As such, MTBE health risks from existing pathways were found to be irrelevant for receptors at the adjacent school.
South Africa

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21

Butters, Terry Douglas. "Ligand-affinity chromatographic purification of Î±-fucosidases and Î±-glucosidases : tools for the structural characterisation of N-linked oligosaccharides from diverse species." Thesis, Open University, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296614.

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22

Marichal-Gallardo, Pável Alejandro Verfasser], and Udo [Gutachter] [Reichl. "Chromatographic purification of biological macromolecules by their capture on hydrophilic surfaces with the aid of non-ionic polymers / Pável Alejandro Marichal-Gallardo ; Gutachter: Udo Reichl." Magdeburg : Universitätsbibliothek Otto-von-Guericke-Universität, 2019. http://d-nb.info/1219965022/34.

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Marichal-Gallardo, Pável Alejandro [Verfasser], and Udo [Gutachter] Reichl. "Chromatographic purification of biological macromolecules by their capture on hydrophilic surfaces with the aid of non-ionic polymers / Pável Alejandro Marichal-Gallardo ; Gutachter: Udo Reichl." Magdeburg : Universitätsbibliothek Otto-von-Guericke-Universität, 2019. http://nbn-resolving.de/urn:nbn:de:gbv:ma9:1-1981185920-332329.

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Marlot, Léa. "Développement de méthodes bidimensionnelles préparatives CPCxLC : application à la purification de molécules d'intérêt issues de matrices végétales." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1299/document.

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La chromatographie bidimensionnelle préparative suscite de plus en plus d'intérêt dans l'élucidation d'échantillons complexes car elle permet de collecter un grand nombre de molécules à haute pureté et quantitée récupérée. Bien que la chromatographie liquide (LC) soit souvent choisie en deuxième dimension, la chromatographie de partage centrifuge (CPC) à de multiples avantages qui en font une technique de choix pour la première dimension. Dans le but de purifier plusieurs molécules d’intérêt dans les matrices végétales, le couplage «comprehensive» CPCxLC représente une technique à fort potentiel. Après avoir expliqué son intérêt et les enjeux liés à la séparation préparative en mode «comprehensive», le développement d’une telle séparation est étudiée selon trois axes. Tout d’abord, une purification de deux molécules d’intérêt dans la plante Edelweiss est réalisée à l’échelle industrielle grâce à la réalisation de cartographies 2D au laboratoire. Cette application permet de montrer l’intérêt du couplage et de mettre en évidence les verrous liés aux conditions de transfert total des fractions en deuxième dimension. Dans une deuxième partie, la séparation CPCxLC en mode « comprehensive » est développée avec le transfert total de l’échantillon en deuxième dimension pour la purification de cinq composés cibles présents dans la plante Edelweiss. Les points clés de la séparation CPCxLC, à savoir le temps d’échantillonnage et le transfert en deuxième dimension, sont étudiés au regard du couplage LCxLC afin de garantir une qualité de séparation permettant la récupération totale des composés. Enfin, la troisième partie consiste à la mise en place d’une méthodologie de sélection des systèmes CPCxLC basée sur l’évaluation quantitative du potentiel des systèmes bidimensionnels à apporter de la distance entre les pics. Cette procédure de sélection est développée sur l’échantillon Cyclopia genistoides avec l’objectif d’isoler huit composés cibles
Preparative two-dimensional chromatography is gaining interest in the elucidation of complex samples as it allows the collection of a large number of molecules with high recovered purity and quantity. While the second dimension is often selected to be liquid chromatography (LC), centrifugal partition chromatography (CPC) is a technique with multiple advantages representing a suitable first dimension. In order to purify several molecules of interest in plant matrices, the comprehensive CPCxLC represents a technique with high potential. After explaining its interest and the issues related to the preparative separation in comprehensive mode, the development of such a separation is studied according to three axes. Firstly, a purification of two targeted molecules in Edelweiss plant is carried out at industrial scale thanks to the realization of 2D-contour plot. This application allows to expose the interest of the separation and to highlight the locks related to the conditions of total transfer of the fractions in second dimension. In a second part, the comprehensive CPCxLC separation is developed with the total transfer of the sample in second dimension applied to the purification of five target compounds from Edelweiss plant. The key points of the CPCxLC separation, namely the sampling time and the second dimension transfer, are studied with regard to the LCxLC separation in order to ensure a separation quality allowing the total recovery of the compounds. Finally, the third part consists in the implementation of a CPCxLC system selection methodology based on the quantitative evaluation of the potential of two-dimensional systems to generate distance between peaks. This selection procedure is developed on the sample Cyclopia genistoides with the objective of isolating eight target compounds

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Bouiche, Feriel. "Amélioration instrumentale de la chromatographie de partage centrifuge en vue de la purification de molécules très polaires." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1003/document.

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L'objectif de cette thèse est de développer un nouvel instrument de chromatographie de partage centrifuge (CPC) dédié à la purification de molécules très polaires. La CPC est une technique préparative permettant la séparation des molécules grâce à l'utilisation d'un système solvant constitué de deux liquides non miscibles. Ce manuscrit expose dans un premier temps les différentes techniques de purification de protéines utilisées dans le cas d'un procédé industriel de production. Un focus est réalisé sur l'utilisation de systèmes biphasiques aqueux pour la purification des biomolécules, qui représente un réel avenir dans l'industrie du fait de son faible coût, de sa facilité de montée en échelle et surtout de l'environnement favorable qu'il fournit aux biomolécules. Ainsi en se basant sur les avantages de ces systèmes solvants dits Aqueous Two Phase Systems (ATPS), la CPC pourrait apporter une efficacité supplémentaire permettant de purifier les protéines à moindre coût. Pour pouvoir répondre à cet enjeu industriel, il est nécessaire de développer à la fois des méthodes chromatographiques innovantes et de nouveaux instruments dédiés. En effet, les instruments de CPC actuels ne sont pas compatibles avec les Bonne Pratique de Fabrication du fait de la présence de joints téflons qui empêche la possibilité de stériliser les instruments. La fabrication d'un nouvel instrument monobloc entièrement en titane a été réalisée grâce à la technologie de l'impression 3D pour répondre à cette problématique. L'objet de cette thèse est l'évaluation poussée des performances de cette nouvelle colonne afin de déterminer son applicabilité à la purification des biomolécules. Un focus sera également apporté à l'injection de volumes très faibles d'échantillon afin de faciliter le développement de méthodes
The aim of this thesis is to develop a new centrifugal partition chromatography (CPC) instrument in order to purify highly polar molecules. CPC is a preparative technique for the separation of molecules using a solvent system composed of two immiscible liquids. This manuscript describes the different protein purification techniques used in industrial production process. A focus is made on the use of aqueous biphasic systems for the purification of biomolecules, which represents a real trend in the industry thanks to its low cost, scaling simplicity and especially the favorable environment that it provides to biomolecules. Thus, based on the advantages of these solvent systems known as Aqueous Two Phase Systems (ATPS), CPC could provide additional performances to purify proteins at lower cost. To respond to this industrial challenge, it is necessary to develop both innovative chromatographic methods and new devoted instruments. Indeed, current CPC instruments are not compatible with Good Manufacturing Practices due to the presence of Teflon seals which prevents the possibility of sterilizing the instruments. The manufacture of a new monobloc instrument entirely made of titanium was achieved thanks to the 3D printing technology. The purpose of this thesis is the evaluation of this new column performance in order to determine its applicability to biomolecules purification. A special attention is also provided to the injection of very small sample volumes in order to facilitate method development

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Blanc, Claire-Line. "Conception et optimisation d’un procédé innovant pour la purification d’acides organiques issus de biotechnologie." Thesis, Châtenay-Malabry, Ecole centrale de Paris, 2015. http://www.theses.fr/2015ECAP0008.

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Le but de cette étude est d’évaluer l’utilisation de la chromatographie préparative dans le cadre de la conception d’un procédé de purification d’acides organiques. Les acides principalement étudiés sont les acides lactique et succinique. Ils sont produits par fermentation et utilisés depuis longtemps dans l’industrie comme additifs. Ils sont aussi identifiés comme des molécules plateformes très intéressantes pour le développement de la chimie verte, à partir de carbone renouvelable. En particulier, ils constituent des monomères pour l’industrie des bioplastiques. A la différence des utilisations historiques, ce type d’application requière des niveaux de pureté beaucoup plus importants. Ces puretés sont atteintes via des étapes supplémentaires d’extraction liquide-liquide, de distillation et/ou de cristallisation. Nous avons cherché à évaluer si la mise en œuvre de la chromatographie préparative pouvait permettre d’atteindre les spécifications requises. Pour cela, la chromatographie a été étudiée en détails en tant qu’opération unitaire, afin de mieux comprendre les mécanismes de séparation des composés étudiés et les paramètres de mise en œuvre. Deux types de résine ont été principalement utilisés, une cationique forte et une anionique forte. Dans un premier temps, l’étude thermodynamique de l’adsorption de trois acides organiques en solution pure a été réalisée. Elle a révélé un comportement très différent pour les deux résines : l’adsorption sur la résine cationique forte est assez linéaire alors que sur l’anionique forte, elle est fortement non linéaire et suit un modèle de Langmuir. L’influence de la vitesse sur la forme des pics et donc la dispersion pendant la séparation a ensuite été étudiée. Il a été montré que l’efficacité de la colonne diminue linéairement avec la vitesse d’élution, conformément au modèle de Van Deemter. Il a aussi été mis en évidence que la pente de cette droite est la même à l’échelle laboratoire et sur le pilote à une échelle dix fois plus grande. Elle peut ainsi permettre de prévoir l’évolution de l’efficacité de la colonne au changement d’échelle. Des solutions en mélange synthétiques et réels ont été étudiées, afin d’évaluer l’influence sur la séparation des paramètres opératoires, tels que la charge, la concentration de l’alimentation, le pH… 2 Sur la résine anionique, une première modélisation a été effectuée à partir de ces résultats expérimentaux. Elle a permis de mettre en évidence, qu’un mécanisme d’adsorption de type Langmuir ne suffit pas à expliquer la forme et la position des pics. Nous avons supposé qu’un mécanisme d’échange d’ions de la forme dissociée des acides organiques pourrait aussi entrer en jeu. Cet échange aurait un impact important sur la forme et la position des pics, bien que les acides organiques soient très majoritairement sous leur forme neutre. Les séparations mises en évidence à l’échelle laboratoire ont été validées à l’échelle pilote en chromatographie continue ISMB. Il a été montré que la résine anionique permet d’atteindre une plus grande pureté que la résine cationique avec une productivité similaire. Un procédé complet de purification a pu être testé avec de l’acide succinique, mettant en jeu une acidification par électrodialyse bipolaire, une concentration par osmose inverse, une séparation par chromatographie préparative sur résine anionique forte et une décoloration par nanofiltration. Le produit a ensuite été cristallisé afin de se comparer à un produit industriel. Le produit obtenu est proche des spécifications attendues et est plutôt meilleur que le produit industriel. Une étape supplémentaire d’échange d’ions aurait vraisemblablement permis d’obtenir des cristaux de grade polymère. Nous avons donc montré que la chromatographie a sa place dans un procédé de purification d’acides organiques, dans le but d’obtenir une très haute pureté
The objective of this study is to evaluate the use of preparative chromatography in the context of the elaboration and optimization of an innovative purification process of organic acids from biotechnology. Lactic and succinic acids were mainly studied. They are produced by fermentation and used in industry as additive, for a long time. They are identified as promising building blocks for green chemistry development, from renewable carbon. In particular, they are monomers for bioplastic industry. Unlike historical utilizations, this new type of application requires much higher purity levels. Those purities are currently obtained by additional purification steps, like liquid-liquid extraction, distillation and/or crystallization. We tried to evaluate if the required specifications may be reached by the implementation of preparative chromatography. For this chromatography was studied in details as unitary operation, in order to better understand separation mechanisms of studied compounds and implementation parameters. Two resin types were mainly used, a strong cationic one and a strong anionic one. Firstly, thermodynamic study of the adsorption of three organic acids in pure solution was performed. It revealed very different performances for both resins: adsorption on strong cationic resin is quite linear, whereas on strong anionic one adsorption is strongly nonlinear and fits with Langmuir model. Elution velocity influence on peak shape and so on dispersion was then studied. Column efficiency decreases linearly with elution velocity, accordingly to Van Deemter model. It was shown that the line slope was identical at lab scale and on a pilot ten times bigger. Then it may be used to predict column efficiency evolution during scale-up. Mixing solutions from synthetic or real origin were studied, to evaluate operational parameter influence on the separation, as load, feed concentration, pH… On the strong anionic resin, a first modeling was developed for experimental results. It highlighted that Langmuir type adsorption mechanism is not able to explain peak shape and position. We supposed that an ion exchange mechanism with the organic acid dissociated part may happen. This exchange may have a significant impact on peak shape and position, even if organic acids are mainly in molecular form, because of a low work pH. 4 Separations established at lab scale were validated at pilot scale in continuous chromatography ISMB. It was demonstrated that the anionic resin allows to reach a higher productivity than the cationic one, with a similar productivity. A complete purification process was tested with succinic acid, using bipolar electrodialysis acidification, reverse osmosis concentration, preparative chromatography separation with a strong anionic resin and nanofiltration discoloration. Product was then crystallized, to be compared to an industrial product. Our crystals were close to waited specifications and relatively better than the industrial ones. An additional ion exchange step could have allows to reach polymer grade. We show that chromatography is useful in an organic acid purification process, in order to reach a very high purity

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27

Forrer, Nicola. "Antibody purification with ion-exchange chromatography." Zürich : ETH, 2008. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17784.

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Ferreira, Filipe Miguel Garcia. "Antibodies purification using centrifugal partition chromatography." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22486.

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Mestrado em Bioquímica - Métodos Biomoleculares
A dificuldade em desenvolver antibióticos mais eficientes, numa altura em que a resistência microbiana tem vindo a aumentar, torna essencial o desenvolvimento de terapias alternativas, económicas e eficazes. Os anticorpos obtidos a partir da gema de ovo de galinha, imunoglobulina Y (IgY), têm-se destacado não só pela sua produção mais simples e em maior quantidade em relação aos anticorpos policlonais de mamífero, mas também devido às inúmeras vantagens em termos de aplicações. No entanto, atualmente não existe uma plataforma de purificação de IgY que seja económica, eficaz e passível de aplicação a nível industrial - uma lacuna que este trabalho se propõe a resolver. Assim, neste trabalho, estudou-se a possibilidade da utilização de sistemas aquosos bifásicos (SAB) compostos por PEG 1000 e tampão fosfato (K2HPO4/KH2PO4) ou K2HPO4, seguidos de um passo de ultrafiltração, ou acoplados à tecnologia de cromatografia de partição de força centrífuga (CPC), para a purificação de IgY. Foram avaliados os efeitos de pH (5,5; 6,0; 6,5; 7,5; e 8,0) e composição de PEG e sal na extração de IgY, bem como as condições utilizadas na CPC (fluxo da fase móvel, rotação e modo de operação). Foi estudada a estabilidade do anticorpo em soluções aquosas dos componentes utilizados nos SAB utilizando dicroísmo circular, assim como a atividade/estabilidade do anticorpo após o processo de purificação por ELISA, salientando assim o efeito do PEG 1000 na estrutura secundária e na atividade do IgY. O ATPS constituído por 18 % PEG 1000 + 13 % tampão fosfato a pH 6,0 conduz aos melhores resultados em termos de purificação, obtendo-se num único passo de extração uma pureza de IgY de 39 %. Após a aplicação de CPR, obteve-se IgY com um grau de pureza de 51 %, e com ultrafiltração, IgY com um grau de pureza de 47 %. Face aos resultados obtidos, destaca-se a CPR como a técnica mais adequada dado que permite obter IgY com um maior grau de pureza e ser passível de aplicação à escala industrial.
The difficulty in developing more effective antibiotics, at a time where the microorganism’s resistance to them has been increasing, turns essential the development of cheaper and effective alternative therapeutics. Antibodies obtained from the chicken’s egg yolk, immunoglobulin Y (IgY), have stood out because of their production simplicity and production in higher quantity when compared to mammal polyclonal antibodies, and also because of their advantages in terms of applicability. Nonetheless, there is still no low-cost, effective and scalable platform for the IgY purification - a vacuity that this work aims to solve. Therefore, in this work, the possibility of using aqueous biphasic systems (ABS) composed of PEG 1000 and phosphate buffer (K2HPO4/KH2PO4) or K2HPO4, followed by an ultrafiltration step, or coupled with centrifugal partition chromatography (CPC), was studied. The effect of pH (5.5; 6.0; 6.5; 7.5 and 8.0) and the PEG + phosphate buffer composition in the extraction of IgY was investigated, as well as the conditions to be used in CPC (mobile phase flow rate, rotation, and the operation mode). The stability of the antibody in aqueous solutions of the components used in the ABS formation was studied using circular dichroism, as well as the activity/stability of the antibody after the purification process, by ELISA, primarily outlining the effect of PEG 1000 in the secondary structure and activity of IgY. The ABS composed of 18 wt % PEG 1000 + 13 wt % phosphate buffer at pH 6.0 yielded the best results in terms of purification, achieving a 39 % IgY purity in a single extraction process. After the application of CPC, an IgY purity of 51 % was obtained, and with the ultrafiltration technique a purity of 47 % was obtained. According to these results, CPC appears as the most adequate purification technique due to the higher purity of IgY obtained and possibility of being applied at an industrial level.

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Stevenson, Steven A. "Chromatography and purification of endohedral metallofullerenes." Diss., Virginia Tech, 1995. http://hdl.handle.net/10919/29176.

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At the conception of this research, a separation methodology for obtaining purified metallofullerene [ A_m@C_2n; m = # of metal atoms, A, and C_2n = # of carbons in the surrounding cage] samples was not yet developed. Isolation of these metal-encapsulated fullerenes was strongly desired for characterization of their physical and chemical properties. Predicted applications for these novel species include their use as possible superconductors, catalysts, and non-linear optical devices. However, initial purification efforts have been hindered by several difficulties. These factors include a low abundance (<1 %) in the raw extract, uncertain stability in aerobic environments, coelution of A_m@C_2n with empty-cage fullerenes, and the need for selective chromatographic detection. In this research, these difficulties have been overcome with the development of a continuous-flow, online HPLC-EPR apparatus. Advantages include a selective, non-invasive detector with chromatographic separations being performed in a controlled anaerobic environment. This on-line approach permits the selective detection of only those metallofullerenes with an odd-number of encapsulated atoms. The ability to continually monitor separations of these paramagnetic species ultimately permits the optimization of chromatographic parameters. The methodology developed from this on-line HPLC-EPR approach has ultimately resulted in purified empty-cage...
Ph. D.

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30

Шебедя, Дмитро Сергійович. "Технологія виробництва субстанції моноклональних антитіл, специфічних до білку HBsAg вірусу гепатиту B. Дільниця культивування". Bachelor's thesis, КПІ ім. Ігоря Сікорського, 2020. https://ela.kpi.ua/handle/123456789/41083.

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Дипломний проєкт: 135 с., 26 рис., 12 табл., 13 формул, 90 посилань. Робота присвячена уніфікації та вдосконаленню технології масового напрацювання виробництва моноклональних антитіл у біореакторі, шляхом іммобілізації клітин продуцента на поверхні напівпроникних мембран та перфузійного типу подачі поживного середовища. На основі порівняльних характеристик, основним продуцентом було обрано найбільш стабільний та високо імуногенний штам гібридомних клітин, отриманих шляхом злиття клітин мієломи та спленоцитів мишачого походження. Штам 95E1 характеризується титром 1:1000 у культуральній рідини та ізотопом антитіл Ig G2α. У роботі наведений опис основних етапів технології з наведенням біохімічних характеристик та фізико-хімічних параметрів. Серед конструкцій біореакторів, що використовуються в технології, було обрано мембранний ферментер половолоконного типу, культивування в якому забезпечує високий вихід цільового продукту. На основі наявної моделі біореактору, було проведено технологічні, конструкційні та гідравлічні розрахунки, з метою модернізації технологічних параметрів та виробничих показників.
Diploma work: 135 p., 26 figures, 12 tables, 13 formulas, 90 references. The work is devoted to the unification and improvement of the technology of mass production of monoclonal antibodies in the bioreactor, by immobilization of producer cells on the surface of semipermeable membranes and perfusion type of nutrient medium. Based on comparative characteristics, the main producer was selected by the most stable and highly immunogenic strain of hybridoma cells obtained by fusion of myeloma cells and splenocytes of mouse origin. Strain 95E1 is characterized by a titer of 1: 1000 in the culture fluid and an isotope of Ig G2α antibodies.Presented data describes the main stages of technology with biochemical characteristics and physicochemical parameters. From between of the designs of bioreactors used in the manufacturing technology, based on the index of a high yield of the target product, was chosen membrane bioreactors with hollow fiber cartridges type. In result of using the real model of the bioreactor as prototype, the technological, structural and hydraulic calculations were performed in order to modernize the technological parameters and production indicators.

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Fargues, Claire. "Chromatographie des protéines appliquée à la purification de la pénicilline acylase : Modélisation de la colonne d'adsorption sur un gel d'hydroxyapatite." Vandoeuvre-les-Nancy, INPL, 1993. http://www.theses.fr/1993INPL014N.

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Ce travail concerne la mise au point d'un procédé de purification d'une enzyme, la pénicilline acylase, par chromatographie. Nous avons établi un schéma de purification en trois étapes qui comporte une prépurification du mélange protéique brut et deux étapes chromatographiques: la première, sur support anionique faible, permet d'éliminer par adsorption plus de 90% des protéines contaminantes alors que l'enzyme passe sans se fixer. La deuxième sur support d'hydroxyapatite, termine la purification de l'enzyme, éluée sélectivement. Une étude chromatographique frontale des courbes de percée permet de séparer les mélanges initiaux en trois grandes catégories protéiques, de comportements différents vis-à-vis des supports. Pour dimensionner et optimiser la séparation chromatographique sur gel d'hydroxyapatite, nous avons réalise une étude thermodynamique de l'adsorption de la pénicilline acylase et de deux autres protéines (albumine et hémoglobine bovines). Les isothermes sont bien modélisées par des équations de Langmuir et bi-Langmuir. Une rapide étude cinétique nous permet d'apprécier les résistances au transfert de matière rencontrées par ces protéines. Ces données servent à la simulation de leur adsorption dynamique en colonne séparément et en mélanges, à l'aide d'un modèle incluant entre autre une diffusion homogène dans le grain. Les expériences monoconstituant ont mis en évidence une limitation au transfert intraparticulaire, qui semble moins grande dans le cas des mélanges binaires; cela met en évidence la variation du coefficient de diffusion avec la composition et la dénaturation protéique

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Nourichafi, Nadia. "Purification des immunoglobulines plasmatiques humaines par chromatographie à partir de la fraction II+III de Cohn." Nancy 1, 1993. http://www.theses.fr/1993NAN19429.

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THEOBALD, JEROME Weber Jean-Victor. "LES FULLERENES : PREPARATION ET PURIFICATION PAR CHROMATOGRAPHIE LIQUIDE PREPARATIVE /." [S.l.] : [s.n.], 1995. ftp://ftp.scd.univ-metz.fr/pub/Theses/1995/Theobald.Jerome.SMZ9544.pdf.

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Théobald, Jérôme. "Les fullerènes : préparation et purification par chromatographie liquide préparative." Metz, 1995. http://docnum.univ-lorraine.fr/public/UPV-M/Theses/1995/Theobald.Jerome.SMZ9544.pdf.

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Le thème de ce travail est la recherche des conditions d'obtention des fullerènes et leur purification. Après avoir fait le point bibliographique sur les méthodes actuelles de production sur les hypothèses concernant les mécanismes de synthèse des fullerènes et les méthodes d'analyses usuelles, nous mettons en valeur les paramètres que nous pensons nécessaires pour la formation de ces molécules : la présence simultanée de précurseurs aromatiques et de conditions de températures élevées. Dans la seconde partie, nous confirmons, par l'ablation laser de matériaux carbones divers, le lien entre la composition de cibles irradiées en aromatiques et la nature des fullerènes produits, et testons la capacité de production de cette méthode. Nous proposons des sources nouvelles et originales de fullerènes : la combustion du polystyrène en présence de toluène, et la récupération de fullerènes dans des résidus carbones issus de procédés industriels variés. Les méthodes de fabrication des fullerènes, les nôtres en particulier, conduisent à des mélanges complexes de fullerènes et d'hydrocarbures aromatiques polycycliques (hap). Nous proposons des solutions pour l'extraction, l'élimination (partielle) des hap et la séparation des fullerènes. Dans cette dernière partie, notre soucis a été de mettre au point une méthode chromatographique adaptable à l'échelle préparative. Nous proposons donc des prévisions de production et de prix de séparation des fullerènes à l'échelle préparative
Our purpose was the study of the production and the purification of fullerenes. After summarizing the bibliographic advances in the field of fullerenes production, synthesis mechanisms hypotheses and usual analysis methods, we focuse on the parameters which are expected to step in these molecules discovered recently : the simultaneous presence of aromatic molecules and warmth. In the second part of this thesis, we perform laser ablation experiments on various carbonaceous materials and show the link between the aromatic behavior of the target and the nature of fullerenes. We also propose original fullerenes sources, as polystyrene and toluene combustion or fullerenes recovery in carbonaceous wastes of some industrial processes. The production methods of fullerenes and particularly those presented in this work, lead to complex, mixtures of fullerenes and polycyclic aromatic compounds (PAC). We propose solutions for extraction, pre-purification for partial PAC elimination and separation of fullerenes. We develop a convenient method easy to be scaled-up. We present production forecasts and cost of fullerenes preparative scale separation

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Emanuelsson, Ida, Anna-Karin Jansson, and Katarina Risö. "Characterising of chromatography gels for purification of erythropoietin." Thesis, Högskolan i Borås, Institutionen Ingenjörshögskolan, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:hb:diva-19097.

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Erythropoietin is a human natural hormone which task is to regulate the amount of red blood cells in the body. At Centro de Inmunología Molecular, situated in Havana, erythropoietin is produced by recombinant DNA-technique. The protein is purified through several chromatography steps. Among other things, Centro de Inmunología Molecular uses affinity chromatography and ion exchange chromatography. To both of these chromatographic methods, gel is used as stationary phase. The aim of this study was to investigate and determine parameters for characterising of two gels, this because Centro de Inmunología Molecular have to exchange the gels. The reason for the gel exchange is that the currently used gels will not be manufactured any more. The gel used in the affinity chromatography is Chelating Sepharose Fast Flow and the gel used in the ion exchange chromatography is Q Sepharose Fast Flow. For both of this gels kinetic parameters and isotherm parameters were determined by experiments. The isotherm parameters qmax and Kd were calculated from an adsorption isotherm. To be able to calculate qmax and Kd for both Q Sepharose Fast Flow gel and Chelating Sepharose Fast Flow gel different experiments were made. A kinetic adsorption and an isotherm adsorption were made on each gel. The kinetic adsorption was made in due to find out how long the two different processes were supposed to run and to understand which part of the mass transfer that is controlling the rate. There is no use to let the process to be in progress any longer than until the adsorption ceases. For the Q Sepharose Fast Flow gel this was after 200 seconds. The adsorption with the Chelating Sepharose Fast Flow gel never ceased completely, but after 1000 seconds the adsorption was so slow that it would be no use to continue the process. If the processes continue after the calculated times only money, hours and recourses will be wasted. The data that were achieved was plotted in two different isotherm adsorption models both the Freundlich- and the Langmuir model, this to determine which model that had the best fit. One could see that the Q Sepharose Fast Flow gel was following the model of Langmuir better and because of this the Langmuir equation was used to calculate qmax and Kd. The qmax for the Q Sepharose Fast Flow gel agreed a lot with the value that Centro de Inmunología Molecular had assumed. When it came to the Chelating Sepharose Fast Flow gel, the same kind of plotting was made. But one could see that this time the data was following the model of Freundlich much better. Therefore a calculation of the desired qmax was impossible. Only the value of Kd was calculated. Because the company Centro de Inmunología Molecular still needed the value of qmax an assumption that the gel was following the model of Langmuir was made. qmax was calculated but without any satisfied results. The programs Excel, Statgraphic and Matlab have been used in all calculations.
Uppsatsnivå: C

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Amarouche, Nassima. "Développement de nouvelles méthodologies en Chromatographie de Partage Centrifuge (CPC) : Application à l’isolement et la purification des peptides pharmaceutiques." Thesis, Reims, 2013. http://www.theses.fr/2013REIMP205.

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Les travaux de cette thèse portent sur le développement de nouvelles méthodologies de purification des peptides pharmaceutiques par chromatographie de partage centrifuge (CPC) dans le but de l'introduction de cette technique comme outil de R&D mais surtout de production en milieu industriel. Le caractère original de ces travaux porte essentiellement sur l'introduction de nouveaux systèmes de solvants et la mise au point de nouveaux procédés de purification en mode CPC co-courant. Les différents aspects liés à l'industrialisation des différents procédés de purification ont également été étudiés.La première partie des travaux a consisté en l'étude de quelques nouveaux aspects de l'intérêt de l'application du mode co-courant en chromatographie de partage centrifuge. Une méthodologie originale de purification des peptides tensioactifs non ioniques en mode CPC co-courant a été mise au point. Cette méthodologie a permis de résoudre les problèmes de perturbations hydrodynamiques et de perte de phase stationnaire engendrés par le caractère tensioactif de ces molécules et a été appliquée avec succès à la purification d'une cyclosporine modifiée douée d'une activité anti-virale et faiblement soluble dans les solvants usuellement utilisés en CLHP. Une étude fondamentale de l'effet du peptide sur le comportement hydrodynamique des deux phases lors de la séparation et la visualisation des modèles d'écoulement au sein de la colonne CPC a permis la mise en évidence du role de la ciclosporine modifiée dans la perturbation de la composition des phases du système chromatographique. D'autres aspects de l'intérêt du mode co-courant en CPC ont été étudiés lors de cette étude, notamment l'amélioration de la robustesse et de la résolution de la séparation.La seconde partie des travaux a porté sur le développement de nouveaux systèmes biphasiques de solvants particulièrement adaptés à la purification des peptides hydrophobes non-ioniques, notamment les intermédiaires de synthèse protégés, qui sont très faiblement solubles dans la plupart des solvants communs utilisés en chromatographie. Deux gammes quaternaire et quinaire de systèmes biphasiques de solvants, ainsi qu'un système biphasique ternaire ont été introduits. L'originalité de ces systèmes porte sur l'usage de solvants verts à fort caractère solvatant tel que le Methyl-THF et le cyclopentyl methyl ether (CPME). Les systèmes développés ont été efficacement utilisés pour la purification en CPC d'une exénatide protégée de 39 acides aminés et d'un peptide protégé de 8 acides aminés intermédiaire de la synthèse de la bivalirudine. Ces systèmes devraient être utiles pour une utilisation générale en CPC pour la séparation des peptides synthétiques hydrophobes libres ou protégés
The work presented in this thesis deals with the development of new methodologies for the purification of pharmaceutical peptides by centrifugal partition chromatography (CPC) in order to introduce this technique as a tool for R & D but also in industrial production. The original character of this work relies on the introduction of new solvent systems and the development of new purification processes based on the co-current CPC mode. The different aspects of the process intensification and industrialization have also been studied.In the first part of the work, a study of some new aspects of the interest of the application of the stationary phase co-current mode in CPC is described. An original method for the purification of non-ionic tensioactive peptides in the co-current CPC mode was developed. This method has been successfully applied to the purification of a modified cyclosporine showing a therapeutic interest. This particular elution mode, taking advantage of the liquid nature of the stationary phase, appears to be an efficient solution to get round some hydrodynamic instabilities that sometimes appears during a purification intensification by CPC. A fundamental study of the effect of the peptide on the hydrodynamic behavior of the two phases in the separation and visualization of flow patterns within the CPC column allowed highlighting the role of the peptide in the disruption of phases composition of the chromatographic system. Other aspects of the interest of the co-current mode in CPC were investigated in this study, including the improvement of the efficiency and the resolution of the separation.The second part of the work focused on the development of new biphasic solvent systems particularly suitable for the purification of hydrophobic non-ionic peptides, including protected intermediates, which are very poorly soluble in the most common solvents used in chromatography. Two new scales of biphasic solvent systems showing a wide range of polarity and a ternary biphasic system were introduced to overcome solubility problems often encountered with synthetic hydrophobic protected peptides. The originality of these systems relies on the use of green solvents with high solvating character such as Methyl-THF and cyclopentyl methyl ether (CPME). The developed systems have been effectively used for the purification in CPC of a 39mer protected exenatide and and a 8mer protected peptide intermediate of bivalirudin synthesis

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37

Seddas, Abdessamad. "Purification du mycoplasma-like organism (mlo) de la flavescence dorée de la vigne par immunoaffinité. Intégrité physique et biologique. Etude des principaux constituants." Dijon, 1994. http://www.theses.fr/1994DIJOS024.

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Les mlo demeurent a ce jour non cultivables, et sont difficiles à extraire dans un bon état de pureté et de conservation a partir de leurs hôtes (plante et insecte vecteur). Le mlo de la flavescence dorée de la vigne (fd-mlo) est expérimentalement maintenu au laboratoire dans la fève vicia faba et la cicadelle euscelidius variegatus. La première partie de la thèse a consisté à mettre au point une méthode de purification des mlo par immunoaffinite à l'aide des anticorps monoclonaux. Nous avons utilisé comme ligand un anticorps monoclonal spécifique (51b5) possédant une grande affinité. L'anticorps a été lie par une liaison covalente sur une matrice synthétique d'hydrazide avid gel (bioprobe), les sites actifs des igg sont orientes vers l'extérieur, les anticorps étant lies par leurs extrémités fc. La meilleure élution des mlo a ete obtenue avec un tampon 0,1 m glycine, ph 11,5. L'observation sur la grille au microscope électronique des mlo purifiés à ete possible par dépôt direct des gouttes de l'eluat sur un film de formwar. Les mlo immunopurifiés sont en bon état d'intégrité physique. Un test d'infectivité a ensuite été réalisé pour tester la pathogénicité des mlo purifié. Ainsi, ces mlo ont été injectes dans l'abdomen des cicadelles saines (acquisition artificielle). Les cicadelles ont été déposées sur des plantes saines (v. Faba). Apres 23 jours d'incubation, ces plantes ont montré des symptômes de la flavescence dorée. Les cicadelles injectées et les plantes inoculées ont donné des réponses positives en elisa avec des anticorps anti fd-mlo. Les protéines des mlo purifies ont été analysées par sds-page. Les profils d'immunorevelation avec un anticorps polyclonal anti fd montrent deux bandes majeures dont les masses moléculaires sont voisines de 55 kda et de 19 kda respectivement. Différents anticorps monoclonaux anti fd ne révèlent que l'une ou l'autre de ces deux bandes majeures. La caractérisation biochimique des antigènes majeurs a été abordée. L’Adn extrait à partir des mlo purifie apparait exempt d’Adn contaminant issu de l'hôte. Cet Adn a ensuite été cloné dans un vecteur plasmidique. Nous avons sélectionné parmi les plasmides recombinants deux inserts d’Adn qui ont été utilises avec succès comme sonde pour détecter les mlo dans les insectes et les plantes malades.

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Brouchon, Julie. "Chromatographie cellulaire d'affinité : étude expérimentale des mécanismes de capture spécifique et implications pour un développement industriel." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066335/document.

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Cette thèse a pour objectif le développement d'une technologie de tri cellulaire reposant sur le principe de la chromatographie d'affinité, pour des applications médicales. Pour cela les mécanismes gouvernant la capture spécifique des cellules dans une colonne chromatographique ont été étudiés. Dans un premier temps une étude sur un système modèle met en évidence le rôle prépondérant de l'étape de transport permettant la rencontre entre les cellules et la surface de capture. Le transport est principalement assuré par la sédimentation des cellules. Cela implique que la vitesse d'écoulement dans la colonne est un paramètre déterminant pour optimiser cette étape de rencontre. La capture de cellules est ensuite étudiée dans son ensemble : la rencontre et la formation du lien spécifique. Selon la vitesse d'écoulement deux régimes se distinguent. Pour des vitesses inférieures à 10-3 m.s-1 la cinétique de capture est gouvernée par la cinétique de rencontre. Pour des vitesses plus élevées toutes les rencontres n'aboutissent pas à une capture spécifique. La cinétique de capture est alors limitée par le transport et par la formation du lien. Cette étude expérimentale permet de dimensionner la technique de tri par chromatographie selon les besoins de l'application considérée. En particulier le tri à grande échelle (jusqu'à 1012 cellules) par chromatographie d'affinité est envisageable en ce qui concerne la durée de séparation et les dimensions de la colonne
Development of cell sorting system based on affinity chromatography for medical application is the goal of this thesis. We study mechanisms responsible for specific cell capture in chromatographic column. First an experimental study on system model emphasize the importance of the transport step, which allows the encounter between cells and surface of capture. Cell sedimentation in the main way of transport. That means flow velocity is a key parameter to optimize this transport step. Then the whole cell capture is studied : encounter and specific link formation. Depending on the flow velocity, there are two regimens. Until a velocity of 10-3 m.s-1, kinetics of capture is governed by encounter kinetics. For higher velocity, only fraction of encounters leads to cell capture. The capture kinetics depends not only on transport, but also on kinetics of formation of specific link. This experimental study allows us to design cell affinity chromatography depending on the need of each application. Especially, cell sorting at industrial scale is conceivable concerning the separation duration and column dimensions

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39

Pezzini, Jérôme. "La chromatographie en mode mixte pour la purification de protéines recombinantes à visée santé : caractérisation des interactions impliquées dans les supports de chromatographie HyperCel®, modélisation et applications." Thesis, Bordeaux 2, 2011. http://www.theses.fr/2011BOR21885/document.

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La chromatographie mode mixte représente l’une des plus grandes évolutions de ces dernières années dans le domaine des bioséparations. Cette technique repose sur l'intervention de plusieurs types d'interactions au sein d'un seul et même support. Les résines de chromatographie mode mixte HEA, PPA et MEP HyperCel portent des groupements aliphatiques, aromatiques, thiophiliques ainsi que des groupements aminés protonables en différentes positions. Au moyen d’expériences de chromatographie, à l’aide de protéines standards aux propriétés spécifiques et de mélanges complexes, nous avons isolé ces différentes interactions. Nous avons mis en évidence l’intervention majeure d'au moins deux types d'interactions au sein de ces supports : interactions hydrophobes et électrostatiques. Nous avons pu observer le comportement des résines lors de variations de pH, de force ionique, de types de sels et de tampons ou lors de la présence d'autres composés organiques. Nous avons mis en évidence l'intervention combinée de ces types d'interactions lors des différentes phases de chromatographie. Le comportement des résines mode mixte a révélé des sélectivités particulières et dont le contrôle ciblé à l'aide de l'environnement a permis le développement de méthodes de purification efficaces et originales. Nous avons pu ainsi développer des applications telles que la purification de fragment d’anticorps (Fab’2) à partir de culture de cellules d’insectes, la capture de protéine de type MBP à partir d’extrait bactériens et la capture d’anticorps monoclonaux à partir de cellules de mammifères (CHO), et ainsi améliorer les conditions d’utilisation de la chromatographie en mode mixte
Mixed mode chromatography is the most innovative technique for bioseparation. Mixed mode resins, as the term suggest, involves multiples types of interaction at the same time. HyperCel mixed mode resins, HEA, PPA and MEP, involve aliphatic, aromatic or thiophilic groups as well as protonable amine located in the spacer arm or as a head group. Using classical chromatographic experiments, standards proteins and complex mixtures, we highlighted the two major types of interactions involved: hydrophobic and electrostatic interactions. We specifically influenced these interactions by modifying the environment in terms of ionic strength, pH, salt types, and other compounds. The combination of these interactions during every phase of a chromatographic process has been demonstrated. Mixed mode resins thus offer unique selectivity that can be controlled by the environment. This allowed us to develop several applications from antibodies fragments capture from insect cells, to the purification of MBP-tagged proteins, through monoclonal antibody capture from CHO cells. We thus enhanced mixed mode chromatography

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40

Yu, Xiao-Jie. "Chromatographie liquide d'affinité de la thrombine sur résines de polystyrène modifié." Paris 13, 1987. http://www.theses.fr/1987PA132003.

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41

Castiblanco, Adriana P. "Expression and Purification of Engineered Calcium Binding Proteins." Digital Archive @ GSU, 2009. http://digitalarchive.gsu.edu/chemistry_theses/20.

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Previous studies in Dr. Yang’s laboratory have established a grafting, design, and subdomain approach in order to investigate the properties behind Ca2+-binding sites located in Ca2+-binding proteins by employing engineered proteins. These approaches have not only enabled us to isolate Ca2+-binding sites and obtain their Ca2+-binding affinities, but also to investigate conformational changes and cooperativity effects upon Ca2+ binding. The focus of my thesis pertains to optimizing the expression and purification of engineered proteins with tailored functions. Proteins were expressed in E. coli using different cell strains, vectors, temperatures, and inducer concentrations. After rigorous expression optimization procedures, proteins were further purified using chromatographic and/or refolding techniques. Expression and purification optimization of proteins is essential for further analyses, since the techniques used for these studies require high protein concentrations and purity. Evaluated proteins had yields between 5-70 mg/L and purities of 80-90% as confirmed by SDS-PAGE electrophoresis.

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42

Boudesocque, Leslie. "Nouvelles méthodologies de purification de peptides par chromatographie de partage centrifuge : application à l'isolement et à la purification de peptides bio-actifs." Reims, 2010. http://theses.univ-reims.fr/sante/2010REIMP205.pdf.

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Les @travaux présentés dans ce manuscrit s'inscrivent dans une optique de valorisation non alimentaire d'une agroressource régionale : la luzerne (Medicago sativa) et plus particulièrement de son pool peptidique. La première partie des travaux a consisté à développer une méthodologie de purification de peptides utilisant la Chromatographie de Partage Centrifuge (CPC) dans un mode de développement particulier, le mode échange d'ions. Une méthodologie de purification par échange d'ions mixte a ainsi mise au point, mettant en jeu deux déplaceurs de façon séquentielle. Par ailleurs, la nature de la phase stationnaire est modulable : homogène ou divisée en deux segments correspondant à un état d'activation différent de l'échangeur. Une étude fondamentale du comportement diffusionnel de l'échangeur dans le système de solvants par RMN DOSY a permis de conforter l'hypothèse de la formation de paires d'ions. L'application du procédé par échange d'ions mixte, que ce soit pour la capture d'un peptide bioactif au sein d'une matrice complexe, ou pour le polissage d'un peptide d'intérêt pharmaceutique, a été réalisée avec succès. Le fractionnement de co-produits issu de la filière industrielle de déshydratation de luzerne a généré des fractions qui ont ensuite été évaluées biologiquement en tant qu'agent antivieillissement potentiel. La purification d'une ciclosporine modifiée à visée anti-VIH est également présentée dans ces travaux. La seconde partie des travaux a porté sur l'évaluation du potentiel des extraits de luzerne comme agent anti-âge cutané, à visée dermo-cosmétique. Le vieillissement cutané est sous la dépendance de nombreux phénomènes moléculaires initiés en partie par des radicaux libres, et amplifiés par l'activation d'une classe particulière d'enzymes : les métalloprotéinases matricielles (MMPs). Le potentiel antioxydant ainsi que l'activité inhibitrice vis-à- vis des MMPs ont été alors évalués. Un extrait brut s'est alors démarqué par son activité antioxydante non-négligeable et une activité inhibitrice prometteuse vis-à-vis d'enzymes clés du vieillissement cutané : les MMP-1 et -3
@This work presented in this manuscript deal with the valorization of alfalfa (Medicago sativa) peptide pool. In the first part of the work, the development of a new methodology for peptide purification using Centrifugal Partition Chromatography (CPC) is described. This process uses the ion exchange mode. A versatile methodology for peptide purification using a new mixed ion exchange mode was developed. A homogeneous or divided, with different exchanger activation rates, stationary phase can also be used. A fundamental study of the diffusion behavior of the exchanger in the solvent system, by NMR DOSY, confirmed the hypothesis of the ion pairs. The application of the mixed ion exchange process, whether for the capture of a bioactive peptide within a complex matrix, or for polishing a peptide of pharmaceutical interest, was successful. Fractionation of alfalfa's extracts generated fractions which were then biologically evaluated as an anti-ageing agent. The purification of an anti-HIV modified cyclosporine is also presented in this work. The second part of the work focused on alfalfa extracts screening as anti-ageing agent. Skin ageing is dependent on many molecular events initiated in part by free radicals, and amplified by activation of a particular class of enzymes: the Matrix Metalloproteases (MMPs). The potential antioxidant and inhibitory activity towards MMPs were then evaluated. A crude extract demonstrates significant antioxidant activity and inhibitory activity towards key enzymes of skin ageing: the MMP-1 and -3

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43

Andersson, Mikael. "Characterisation of Chromatography Media Aimed for Purification of Biomolecules." Doctoral thesis, Uppsala universitet, Analytisk kemi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-234743.

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Chromatography media (resins) are very important for and widely used by the biopharma industry in large scale production of biopharmaceuticals, e.g. monoclonal antibodies. Today there are several hundred biopharmaceuticals released globally on the healthcare market. This thesis discusses various strategies and methods for the characterisation of chemical and functional stability of chromatography media. In addition, various analytical techniques used in these areas were evaluated and applied. Further, more specific physical and chemical characterisation methods were evaluated and applied to explore different properties of various chromatography media. In Papers I-III, established methodologies for performing chemical and functional stability studies were used. Mainly agarose-based chromatography media were investigated. For fast screening of the chemical stability, the total organic carbon analysis technique was evaluated and applied. This technique that measures the carbon leakage from the chromatography media at different conditions, proved to be very suitable and robust. For detection and/or identification of leakage compounds responsible for or for part of the measured carbon leakage, different methods such as (high performance) liquid chromatography and gas chromatography mass spectrometry were used. In Papers IV-VII, different properties (i.e. functional performance, ligand content and surface chemistry) were evaluated for different agarose-based chromatography media. Standard chromatographic methods (ion exchange chromatography) and spectroscopic methods (e.g. Fourier transform infrared spectroscopy and time-of-flight secondary ion mass spectrometry) were evaluated and applied. Chemometric methods were used for efficient evaluation of data. Information of chemical, functional and leakage data of chromatography media are valuable and important for the biopharmaceutical companies to be able to fulfil the regulatory requirements of biopharmaceuticals. In addition, information of various chemical, functional and physical properties of chromatography media is likewise important during development and set up of new biopharmaceutical processes.

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44

Hey, Carolyn McKenzie. "Antibody Purification from Tobacco by Protein A Affinity Chromatography." Thesis, Virginia Tech, 2010. http://hdl.handle.net/10919/42645.

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Antibodies represent the largest group of biopharmaceuticals. Due to the nature of their clinical applications, they often need to be produced in large quantities. Plants have distinct advantages of producing large quantities of recombinant proteins, and tobacco is arguably the most promising plant for plant-made-pharmaceuticals (PMP) due to its high biomass yields and robust transformation technology. However, to produce proteins using transgenic tobacco for human applications, purification of the proteins is challenging. On the other hand, Protein A, a bacterial cell wall protein isolated from Staphylococcus aureus that binds to the Fc regions of immunoglobulins, is useful to the isolation and purification of antibodies. An affinity chromatography purification step utilizing Protein A resin introduced early in the purification process can reduce successive unit operations, thereby reducing the overall process cost. However, directly applying tobacco extract to Protein A chromatography columns may be problematic due to the non-specific binding of native tobacco proteins (NTP). In this project, three different Protein A resins, ProSepvA High Capacity, ProSep-vA Ultra, and ProSep Ultra Plus, marketed by Millipore, were studied to provide valuable information for future downstream processes for antibody purification from transgenic tobacco. The efficiency of the post load wash buffer to reduce non-specific binding of NTP to the ProSep A resins were evaluated by altering the ionic strength and pH. Lower salt concentrations of sodium chloride (NaCl) in the post load wash preformed best at reducing the non-specific binding of NTP to the ProSep A resins, while higher salt concentrations were more effective at reducing the amount of NTP contaminants present during elution of the columns. Using a post load wash buffer with an intermediate pH between the binding buffer and the elution buffer was more efficient at eluting our model antibody, human IgG. However, lowering the ionic strength and the pH of the post load wash buffer resulted in a greater presence of IgG prematurely eluting from the ProSep A resins. The non-specific binding of NTP to the resins reduced the dynamic binding capacity (DBC) of the resins after repeated cycles of tobacco extract samples were loaded onto the column. Nevertheless, cleaning the columns with denaturing solutions, such as urea or guanidine hydrochloride, every 8-10 cycles was effective in regenerating the DBC of the resins and prolonging the life cycle of the resins. This is important to evaluating the economic feasibility of directly using Protein A chromatography to recover antibodies from tobacco extract. Of the three Protein A resins studied, ProSep Ultra Plus performed best for antibody purification from tobacco using a PBS wash buffer with a lower ionic strength of 140mM NaCl and an intermediate pH of 5.
Master of Science

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45

Fernando, Samantha. "Monoclonal antibody (mAb) purification by counter current chromatography (CCC)." Thesis, Brunel University, 2011. http://bura.brunel.ac.uk/handle/2438/6522.

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Counter current chromatography (CCC) is a form of liquid liquid chromatography, which the Brunel Institute for Bioengineering (BIB) team have developed to process scale. In this thesis, its application has been successfully extended to the rapid, scalable purification of monoclonal antibodies (mAb) from mammalian cell culture, using aqueous two-phase systems (ATPS) of inorganic salts and polymer. A polyethylene glycol (PEG) and sodium citrate system was found to be the most appropriate by robotic phase system selection. The search for an economical alternative to protein A HPLC is a substantial bioprocessing concern; in this work CCC has been investigated. Initial studies showed that unpredictably, despite separation from impurities being achieved, some loss in the IgG‘s ability to bind to Protein A was seen, as confirmed by Protein A BiaCore analysis. CCC machines were seen to adversely affect IgG functionality. This led to a systematic investigation of the effect of CCC phase mixing on IgG functionality in a number of different CCC instruments, allowing direct comparisons of modes of CCC (hydrodynamic and hydrostatic CCC) and their associated mixing (wave-like and cascade, respectively). The varying g forces produced within the CCC column were determined using a recently developed model to calculate g force range. The effect of interfacial tension was also studied using a custom built 'g' shaker. The optimum CCC mode was identified to be the non synchronous CCC, operated in a hydrodynamic mode but allowing bobbin to rotor speed (Pr ratio) to be controlled independently. In a normal synchronous J type centrifuge a Pr of 1 is fixed, this is where the bobbin and rotor speed are identical I.e. one bobbin rotation (where mixing occurs) to one rotor revolution (where settling occurs). Constraints were seen with this 1:1 ratio and the separation of mAb using ATPS. This work has shown with the use of the non synchronous CCC at a Pr of 0.33, mixing is reduced and rotor rotations increased. Consequently the associated g force range is decreased. Furthermore, by the extension of settling time, the clear separation of the mAb from impurities has been achieved with retention of biological activity. This thesis demonstrates the importance of settling time for ATPS in phase separation and documents the fundamental requirements for the successful separation of biologics. Purified non synchronous CCC samples have additionally undergone rigorous quality control testing at Lonza Biologics by their purification scientists. This work has ultimately showed that with optimisation, the non synchronous CCC can be used to produce biological samples that are of industry standard.

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46

Hsu, Kuang-Hsin. "Scale-up of affinity chromatography for protein purifications." Ohio : Ohio University, 2000. http://www.ohiolink.edu/etd/view.cgi?ohiou1172002240.

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47

Hidayah, Siti Nurul [Verfasser]. "Improvement of liquid chromatography for analysis and purification of proteoforms via rational protein purification parameter screening and sample displacement chromatography / Siti Nurul Hidayah." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2021. http://d-nb.info/1241743037/34.

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48

Ring, Ludwig. "Purification of psychoactive biomolecules in plants using size exclusion chromatography." Thesis, Linköping University, Department of Physics, Chemistry and Biology, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-18434.

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Size exclusion chromatography (SEC) was applied for purification of psychoactive biomolecules from plants. These molecules are in the same molecular weight range, but do not necessarily share other chemical properties, that makes the SEC technique efficient. By applying SEC as a first purification step much of the co-extractives from the plants can easily be removed. Large amounts of target substance can be obtained with little effort if the system is automated. Combining SEC with a second purification step, consisting of normal phase chromatography, provides high purity of the target substance.

Both known and unknown psychoactive biomolecules can easily be purified using the purification method developed in this Master's Thesis. Purifications that previously required long time and much "hands-on" can be completed much faster and with less manual work.

The method developed was tested on cannabis, coffee and 'Spice' with good results.

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49

McElhiney, Jacqueline. "Purification, detection and biological effects of cyanobacterial toxins." Thesis, Robert Gordon University, 1999. http://hdl.handle.net/10059/528.

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The aesthetic beauty of a landscape is a very subjective issue: every person has their own opinions and their own idea of what beauty is. However, all people have a common evolutionary history, and, according to the Biophilia hypothesis, a genetic predisposition to liking certain types of landscapes. It is possible that this common inheritance allows us to attempt to model scenic preference for natural landscapes. The ideal type of model for such predictions is the psychophysical preference model, integrating psychological responses to landscapes with objective measurements of quantitative and qualitative landscape variables. Such models commonly predict two thirds of the variance in the predications of the general public for natural landscapes. In order to create such a model three sets of data were required: landscape photographs (surrogates of the actual landscape), landscape preference data and landscape component variable measurements. The Internet was used to run a questionnaire survey; a novel, yet flexible, environmentally friendly and simple method of data gathering, resulting in one hundred and eighty responses. A geographic information system was used to digitise ninety landscape photographs and measure their landforms (based on elevation) in terms of areas and perimeters, their colours and proxies for their complexity and coherence. Landscape preference models were created by running multiple linear regressions using normalised preference data and the landscape component variables, including mathematical transformations of these variables. The eight models created predicted over sixty percent of variance in the responses and had moderate to high correlations with a second set of landscape preference data. A common base to the models were the variables of complexity, water and mountain landform, in particular the presence or absence of water and mountains was noted as being significant in determining landscape scenic preference. In order to fully establish the utility of these models, they were further tested against: changes in weather and season; the addition of cultural structures; different photographers; alternate film types; different focal lengths; and composition. Results showed that weather and season were not significant in determining landscape preference; cultural structures increased preferences for landscapes; and photographs taken by different people did not produce consistent results from the predictive models. It was also found that film type was not significant and that changes in focal length altered preferences for landscapes.

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Karnchanasri, Kritsadanchalee. "Bi-layered chromatography matrices for the purification of biological nanoplexes." Thesis, University of Birmingham, 2013. http://etheses.bham.ac.uk//id/eprint/4149/.

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The preparations of SEC/IEC supports from commercially available, underivatised base matrices by AGE activation-partial bromination technique via two different approaches; (i) viscosity enhanced-reaction diffusion (VE-RD) and (ii) microwave-assisted reaction diffusion, were studied and optimised. Selected supports produced by both approaches were further evaluated by applying to packed bed chromatography system in order to purify target pDNA from neutralized E.coli cleared lysates.For VE-RD approach, viscosity enhancement by sucrose (< 0.032 Pa.s) was found to greatly aid the creation of thin inert outer layer. The optimum condition for SEC/IEC Sepharose CL-6B production observed was 10% single bromination at room temperature in 64% (w/v) aqueous sucrose without sodium acetate addition. The effects of different base matrices, conductivities, linear flow rate, target pDNA sizes and support preparations on chromatography performance were investigated. Microwave irradiation heated up the reaction in a rapid controlled manner compared to conventional heating. SEC/IEC Sepharose CL-6B produced via microwave-assisted reaction diffusion approach at 80oC with 10% partial bromination showed almost complete surface charge elimination with the highest SI value of 57.4. This support showed the high core binding capacity. However, a delayed pDNA breakthrough was also observed. It was noted that the plasmid forms remain unchanged after SEC/IEC column purification.

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Дисертації з теми "Chromatographic purification"

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