Dissertations / Theses on the topic 'Affinity adsorption'

It is important to understand the processes that regulate phosphorus (P) fluxes to coastal environments, because P is an important nutrient in coastal ecosystems. Phosphorus adsorbs to the surface of minerals in sediment and bedrock, and an influx of seawater can cause some of that P to desorb, raising the P concentration of ambient water. Although seawater-induced P desorption is thought to be an important source of P to coastal environments, the chemical reactions that underlie it have not been established. Previous work provides some relevant surface reactions and associated affinity constants between various aqueous P species and the surface of calcite and in dilute calcium carbonate-P solutions. However, these reactions with their respective affinity constants from the literature fail to predict the behavior of P with calcite in seawater. In this study, we conducted a series of batch experiments involving both adsorption and desorption of P in seawater, freshwater, dilute seawater, and mixtures of seawater and freshwater. We used these results in the geochemical model PHREEQC and the parameter estimation model PEST to optimize the affinity constants for the existing surface reactions. We found that after making minor adjustments to the affinity constants, the existing surface complexation models of calcite surface reactions from the published literature are sufficient to explain seawater-induced P desorption. Specifically, our results suggest that CaPO4- and either CaHPO40 or HPO42- may be important species in the P adsorption/desorption reactions in freshwater-seawater mixing.

A polymer matrix of hydroxy ethyl methacrylate - ethylene glycol dimethacrylate was synthesised and coupled with the ligand p-aminobenzamidine. Although the adsorbent displayed sufficient affinity towards trypsin, the overall adsorption performance was not satisfactory. Subsequent studies were carried out with a p-aminobenzamidine silica adsorbent, which gave far superior results. Amounts of up to 35 mg of trypsin per g of silica could be adsorbed from a porcine pancreatic extract at buffer conditions of 0.05 M Tris, 0.5 M NaCl, pH 8. Complete elution was achieved with a buffer of 0.1 M acetic acid, pH 3. The system was characterised by fixed bed breakthrough studies of the adsorption, wash and desorption step. A method of data analysis was developed and applied to the experimental results. Axial dispersion could be identified as the predominant 'mass transfer' mechanism. A simulation model was build, based on the data analysis results. The model was verified successfully against the experimental data. Continuous separation experiments were performed with three fluidised beds, namely an adsorption, desorption and wash column, with circulation of solids between the columns. Purities of 83% and 66% were achieved in these runs with complete recovery of trypsin. A continuous process model, also based on the data analysis results and on fluidised bed characteristics, was found to agree fairly well with the experimental data. Simulation experiments were conducted to find the optimum configuration of the process. Best performances with respect to product purity were obtained for an adsorption column height of 3 cm and a wash column height of 4 cm. Step experiments revealed that the process is relatively easy to control if the solid flow rate is chosen as the control parameter.

Cellulosic biomass is the major organic matter produced in the biosphere. The biodegradation of this cellulosic material is achieved by enzymatic activities of the cellulose degrading microorganisms. These organisms usually express a complex extracellular or a membrane bound cellulolytic system comprising combination of several cellulase enzymes. Cellulases are the group of hydrolytic enzymes capable of hydrolysing insoluble cellulose to glucose. Phlebia gigantea is an aggressive white rot basidiomycete with ability to tolerate resinous extracts on freshly cut wood and higher growth rate. This helps the fungus to colonise the sapwood preventing other fungi from becoming established. Early research on the cellulase system of this organism reported the presence of a cellulase system composed of P-glucosidase, endoglucanase and a cellobiohydrolase. Based on these unpublished studies, our aim was to obtain a complete sequence of putative cellobiohydrolase I (CbhI) from this organism. Attempts to identify and isolate the cellulase gene resulted in an incomplete cDNA sequence of I 154 bp. To understand the cellulase system, expression and regulation of the cellulase enzymatic activity was examined for incubation of P. gigantea on substrates glucose, xylose, Avicel, carboxymethyl cellulose and cellobiose. The pH, total protein and biomass production results indicated that the capacity of P. gigantea to degrade cellulose is dependent upon the nature of the carbon source and the regulation of the cellulase synthesis is repressed in the presence of simple sugars like glucose and xylose. The study employed the highly effective method of purification by affinity adsorption and purified cellulase complex in large quantity. Characterisation of the kinetic properties of this cellulase complex revealed that the rate of cellulase catalysis were optimum at pH 5.0 and temperature 50GC. The purified complex was comprised of multiple proteins and demonstrated significant CMCase and CBHase activity on zymogram analysis. The purified cellulase complex was characterised by 2D gel electrophoresis and by peptide mass finger printing using MALDI-TOF massspectrometry analysis. The 2D gel analysis of the purified cellulase complex showed 15 spots within the range of pI 3.5 to pI 7 and the molecular weight between 20KDa to 100KDa. Three protein spots were selected based on the IEF and SDS zymogram and identified using MALDI-TOF MS analysis. These proteins were identified based on the peptide mass data belonging to the 6-phospho-a-glucosidase, p-glucosidase and glycosyl hydrolase family 13 a-amylase or pullulanases, suggesting the divergent evolution of specific cellulase proteins. This study showed P. gigantea as a potential cellulase source and the cellulase complex secreted by the induction of substrate, comprises a variety of enzymes related to hydrolysis of cellulose biomass. It is evident from this and previous studies that P. gigantea cellulase complex comprises of a specific set of enzymes that possess the ability to degrade crystalline cellulose and is one of the first organisms to colonise freshly cut wood. Further studies on the cellulase system of this primary colonist may open up the prospects to utilise this organism as the potential onsite bioreactor agent, pre-treating the biomass and increasing the economic feasibility of the industrial bioenergy processes.

In this work, the main emphasis of the research concerns the development of isolation and purification methods of biomolecules from biological fluids. Several separation techniques were incorporated in chromatographic gels to obtain multifunctional hybrid chromatographic separation media for proteins, peptides and amino acid isolation and purification.In the first part of the research, several chelating agents were synthesized and their effectiveness to purify immunoglobulins using Immobilized Metal Affinity Chromatography (IMAC) was investigated. Ethylenediamine triacetic acid (TED) with immobilized copper resulted in the most effective in terms of purification and protein capacities.The next part of the work involved the development of hybrid chromatographic media that combines protein specific adsorption with sharp controlled size access permeation. This was accomplished by incorporating two types of ligand derivatives, one that permits the permeation of only certain molecular size range compounds, and a second one that specifically binds target biomolecules among the compounds of that specific molecular size range. Hybrid systems included binding ligands for Immobilized Metal Affinity Chromatography (IMAC), Ion Exchange Chromatography (IEX) and Hydrophobic Interaction Chromatography (HIC) combined with a controlled access polymer at different densities such as polyethylene glycol (PEG) and dextran derivatives. In general, low grafting density of high molecular weight PEG was found to be as effective as high grafting density of low molecular weight PEG in the rejecting properties of the semi-permeable synthesized media.Theoretical and experimental batch adsorption studies were also performed with the hybrid media and a mathematical model was developed to study the uptake of proteins under specific conditions of controlled permeation.In the last stage of this work, chelating surfactants were synthesized and used as reversible affinity ligands on reversed phase adsorbents for protein separations.One of the main accomplishments of this research was the development of separation media for small molecular size compounds from larger molecules and from complex biological systems. Applications of special interest will include the isolation and purification of solutes, such as metal ions, toxins, drugs, biomolecules, including proteins, biotoxins, nucleic acids, peptides, hormones, and biomarkers from biological fluids (such as human serum, urine, etc.) and from aqueous solutions.

Orientadores : Sonia Maria Alves Bueno, Ricardo de Lima Zollner
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica
Made available in DSpace on 2018-08-06T11:50:08Z (GMT). No. of bitstreams: 1 Duarte_IsaSantos_D.pdf: 749498 bytes, checksum: 83d66753c75e7828fd4a8b858aecf473 (MD5) Previous issue date: 2006
Resumo: A alergia é uma enfermidade do sistema imunológico que afeta aproximadamente de 20 a 30% da população mundial. Dentre as reações alérgicas, a reação de hipersensibilidade imediata é mediada pelas imunoglobulinas E (IgE). Os indivíduos geneticamente predispostos a manifestar reações por hipersensibilidade imediata e polissensibilizados aos alérgenos ambientais são considerados atópicos e, geralmente, possuem teores de IgE total até 10.000 vezes mais elevados do que as pessoas não-atópicas. O conhecimento das interações entre a IgE e ligantes de afinidade pode levar ao desenvolvimento de novos tratamentos da hipersensibilidade imediata, por exemplo a terapia de adsorção seletiva através de circulação extracorpórea, assim como ao desenvolvimento de métodos de obtenção de IgE purificada, para aplicação nas áreas de diagnóstico, pesquisa molecular, dentre outras. Os adsorventes empregados na terapia de adsorção seletiva, bem como na purificação de IgE, geralmente são anticorpos anti-IgE imobilizados em agarose, os quais são de alto custo e difícil obtenção. Este trabalho avaliou o desempenho de adsorventes alternativos ao Sepharose-anti-IgE, visando a remoção de IgE total e específica aos ácaros Dermatophagoides pteronyssinus e Blomia tropicalis de amostras plasmáticas e a preparação de soluções enriquecidas em IgE, como uma das etapas do processo de purificação de IgE. Os adsorventes estudados constituíram-se de lectinas (concanavalina A e Lens culinaris), aminas (poli-L-lisina e aminohexil) e o aminoácido D-triptofano, imobilizados em agarose. Dentre eles, o gel agarose-Lens culinaris mostrou-se o mais promissor para aplicação na terapia de adsorção seletiva de IgE e o gel Sepharose-concanavalina A mostrou-se o mais adequado para ser usado na obtenção de soluções enriquecidas em IgE. Experimentos cromatográficos foram realizados visando estabelecer condições experimentais (velocidade superficial, número de passagens de plasma pela coluna, temperatura e razão entre volume de plasma e volume de leito) mais favoráveis à adsorção de IgE em agarose-Lens culinaris. Posteriormente, essas condições foram utilizadas nos experimentos de simulação in vitro de circulação extracorpórea, nos quais o gel agarose-Lens culinaris removeu de 40,7 a 42,8% de IgE¿s total e específicas. A obtenção da solução enriquecida em IgE foi realizada por meio de duas etapas cromatográficas, empregando-se os princípios de afinidade (colunas agarosejacalina e Sepharose-concanavalina A) e de exclusão por tamanho (permeação em gel). A solução final enriquecida em IgE obtida, continha como principais impurezas, IgA e IgG. Como resultado das duas etapas, 36,6% de IgE foi recuperada e o fator de enriquecimento em IgE, em relação a IgA, IgG, IgM e albumina, foi de 75,8. Apesar do gel Sepharose-anti- IgE apresentar desempenho melhor tanto na remoção quanto na purificação de IgE, os adsorventes agarose-Lens culinaris e Sepharose-concanavalina A apresentam custos mais atrativos
Abstract: Allergy is a disorder of the imune system, affecting approximately 20%-30% of the general population. Among allergic reactions, immediate hypersensitivity is mediated by immunoglobulin E (IgE). Individuals that have a genetic predisposition for responses to immediate hypersensitivity are named atopic and generally have elevated serum IgE concentration, up to 10,000-fold higher than in the normal population. The knowledge of the interactions between IgE and affinity ligands may lead to the development of new methods of treatment for immediate hypersensitivity, for example, IgE selective adsorption therapy through extracorporeal circulation, as well as to new methods for obtaining purified IgE, which is employed in diagnostic and in molecular research. The adsorbents employed in IgE selective adsorption therapy, as well as in IgE purification, are usually antibodies anti-IgE immobilized on agarose, which have high costs and are difficult to obtain. This work assessed the performance of adsorbents (alternative to Sepharose-anti-IgE) for the removal of total IgE and IgE specific for the airbone allergens Dermatophagoides pteronyssinus and Blomia tropicalis from plasma samples, as well as in the production of IgE enriched solutions, considered as a step of IgE purification. The adsorbents studied were lectins (concanavalina A and Lens culinaris), amines (poli-L-lisina e aminohexil) and the aminoacid D-tryptophan, all of them immobilized on agarose. Among them, Lens culinaris-agarose showed the best performance for IgE selective adsorption therapy, and Sepharose¿concanavalin A was considered the most appropriate for the production of IgE enriched solutions. Chromatographic experiments were accomplished in order to determine operating conditions (superficial velocity, number of times the plasma passed through the column, temperature, and ratio of plasma volume to bed volume) more favorable to IgE adsorption on Lens culinaris-agarose. The selected conditions were utilized in in vitro simulation assays of extracorporeal circulation, in which the Lens culinaris-agarose removed from 40.7% to 42.8% of total and specific IgE. The production of IgE enriched solutions was carried out with two chromatographic steps, employing affinity (columns jacalin-agarose and Sepharose-concanavalin A) and size exclusion (gel permeation) principles. The IgE enriched final solution contained IgA and IgG as the major impurities. As a result of both steps, 36.6% of IgE was recovered and the IgE enrichement number concerning IgA, IgG, IgM, and albumin was 75.8. Despite Sepharose-anti-IgE has better performance in removal and purification of IgE, Lens culinaris-agarose and Sepharose-concanavalin A adsorbents have more attractive costs
Doutorado
Desenvolvimento de Processos Biotecnologicos
Doutora em Engenharia Quimica

Etude théorique de l'évolution morphologique du gel de silice (Rhône-Poulenc, Prolabo) sous l'action combinée de la température et de la vapeur d'adsorbat (eau, méthanol). Application à la réfrigération. Le système gel de silice-adsorbat (eau, méthanol) est très efficace dans le cas des faibles différences de température entre la source froide et la source chaude, et la source froide et la température de l'évaporateur; mais il est très sensible aux écarts de température au niveau de l'adsorbeur. La faible vitesse d'adsorption entraine quelques difficultés à utiliser ce système pour la mise en froid d'une charge. Par contre, il sera facilement utilisable pour la compensation des pertes thermiques.

Recherche d'adsorbants synthétiques constitués de polymères fonctionnels capables d'adsorber les anticorps anti viii: c dans des systèmes d'épuration plasmatique. Substitution de fonction sulfonate et sulfamion d'un acide aminés ou de deux acides amines sur un polystyrène

L'utilisation de la sépiolite comme absorbant dans les machines thermiques à affinité est étudiée. Ses propriétés physico-chimiques ainsi que son comportement cinétique dans les systèmes liquide-vapeur-adsorbant sont déterminés. Son comportement dans les machines réfrigérantes à affinité chimique est étudié. Par comparaison avec la zéolite 4a, il apparait que le couple zeolite 4a-H::(2)o est plus apte a la réfrigération que le couple sépiolite-H::(2)o

Des membranes à fibres creuses ont été utilisé pour immobiliser le ligand histidine du fait de leur bon rapport surface membranaire/volume. Les membranes en PEVA se sont avéré le plus compatible. Ainsi, nous avons purifié les IgG sur le support Histidyl-PEVA fibres creuses dans une seule étape, à partir du sérum ou du plasma humain. La meilleure adsorption d'IgG sur ce support membranaire a été obtenue en présence d'ions de tampons zwitterioniques. Nous avons constaté que la sélectivité se manifeste au niveau des pl et des sous-classes des IgG. L'utilisation des tampons zwitterioniques ont permis de séparer la totalité de la fraction IgG du sérum, tandis que d'autres tampons ont permis l'adsorption sélective des IgG1 et des IgG3 ainsi que des IgG avec des points isoélectriques déterminés. Nous avons montré par la suite que l'adsorption des IgG humaine sur l'histidine se fait sur la partie Fab de la molécule. Nous avons également étudié quelques aspects cinétiques et de transfert de matière de ce système d'affinité. Cette étude a été essentielle pour le développement et l'optimisation du procédé de séparation d'IgG. La capacité de notre support en adsorber les IgG a été 3 à 8 fois supérieur à celle de la membrane à fibres creuses couplée à la protéine A décrite dans la littérature. Nous avons également envisagé d'appliquer notre système pour l'élimination des IgG des patients atteints de maladies immunitaires par circulation extracorporelle
Previous work in our laboratory has shown that the amino acid histidine can be used a ligand for the purification of human immunoglobulin subclasses and monoclonal antibodies. Ln this work, we tried to combine the specificity of the ligand histidine with hollow fiber membrane technology to create a powerful device for the separation of IgG for human serum and for other sources. We chose poly( ethylene vinyl alcohol) (PEVA) hollow fibber membranes as the support material. The best IgG adsorption was obtained in presence of the zwitterionic buffers. By choosing the appropriate buffer system, it was possible to adsorb on membrane specifically different IgG subsets. For instance, in Mops buffer, IgG1, IgG2 and IgG3 were bound, whereas in Tris-HC1, only IgG3 and a part of the IgG1 fraction were retained. The parameters involved in the optimization of the separation process, namely the influence of residence time and serum and plasma dilution on immunoglobulin adsorption were studied in view of two applications: purification of IgG and removal of IgG from plasma in clinical aphaeresis (extracorporeal device). The capacity of Histidyl-PEVA calculated on the basis of unit membrane volume was 86 mg IgG/ml. Ln comparation of the protein A-membrane reported in literature, the capacity of Histidyl-PEVA was found to be 3 to 8 fold higher

碩士
國立成功大學
化學工程學系
87
Abstract To maintain the activity of enzymes during separation and purification process is a very important issue. The aim of this research is to use immobilized metal affinity adsorbent on the adsorption of enzymes such as Bacillus licheniformis a-amylase and the a-amylase containing fermentation broth from Bacillus amyloliquefaciens. This sort of adsorbent is made by polysaccharide support, ligand, and metal ion. This research is mainly for the applications of separation and purification. Sepharose 6B and Sepharose 4B were used as the supporting materials for making affinity adsorbents to study the adsorption effect of a-amylases. The chelating results of EDTA and copper ion, EDTA and the copper ion which has formed a complex with Sepharose 6B-IDA were both discussed. Imidazol was used to desorb the enzymes which were adsorbed on the affinity adsorbents. The influence of the imidazol concentration and the operation temperature during desorption stage were also investigated. Concluded from the above, with Sepharose 6B-IDA-Cu2+ to adsorb a-amylases under 37℃ can achieve around 95% activity recovery. The best concentration of imidazol for desorption was 200mM.

The monoclonal antibody market has been growing rapidly in the past decades, and the number of therapeutic areas where monoclonal antibodies (mAbs) are employed has been increasing, with cancer and autoimmune diseases being the most represented. There are already more than 50 approved products, representing a staggering $100 billion in global sales. Because of this high demand, antibody manufacturing has been in constant evolution and asking for new, more efficient, and more optimized methods to be applied both in the upstream and downstream processing. Protein A chromatography is step of choice of most of the pharmaceutical companies for the antibody capture in the downstream processing. It is a core unit operation that has been in constant evolution, with the new resins coming to the market having higher binding capacities than their predecessors and improved alkaline stability. Despite of this extensive improvement in Protein A resins, there are still some aspects that lack understanding and deep investigation, specifically the mechanism of interaction between the antibodies and the Protein A ligands, both under linear and overloaded conditions. This knowledge can be used for further enhancement of performance in the mAbs capture step. The knowledge accumulated during the last decades by studying chromatography for proteins bioprocess development has shed some light to the mechanistic understanding of protein–ligand interactions, though based on indirect measurements. Chromatography processes in general are characterized with online and offline sensors that probe the concentration (UV detector), purity (SEC-HPLC), potency (SPR), and structure (MALS, CD) of the product, as well as conductivity and pH that can be measured directly in the chromatography stations (ÄKTA). However, none of these probes operate in situ, i.e. in the chromatographic column where the interaction occurs. The online sensors tackle the elution peak, and the offline sensors analyse the sample afterwards. Therefore, this research consists in a biophysical study on the antibody adsorption to commercial Protein A resins with in-situ sensors, which resulted in an improved understanding of antibody–Protein A interactions, both under linear and overloaded conditions. Flow microcalorimetry was extensively used to retrieve the thermodynamic parameters during antibody adsorption. The microcalorimeter consists on a ID 6 mm × 6 mm column with two thermistors coupled on the column walls that are able to detect small changes in potential during a chromatographic process. The application of the technique to two commercial Protein A resins (MabSelect SuRe with a tetrameric Protein A ligand and TOYOPEARL AF-rProtein A HC with a hexameric Protein A ligand) showed an adsorption profile of exothermic nature with two sub-processes involved. The first and stronger moment was associated to the adsorption process itself. The second moment, less energetic, was associated either to reorganization of the antibody layer and the Protein A chain upon binding, or to antibody binding to a ligand where an antibody molecule would already be bound. These interpretations were reinforced by the small angle X-ray scattering (SAXS) studies. To characterize the changes in the antibody-Protein-A ligand complex and evaluate the influence of the surface topology on adsorption, SAXS was employed using a miniaturized, X-ray-transparent chromatography column packed with the resin. In this way, the protein absorption process could be followed and the formation of a protein layer on the chromatography resin fibres can be observed at the nanoscale and in a time-resolved manner. For the first time it was possible to directly correlate the nanostructure changes inside the column, upon adsorption and during elution. It was demonstrated the possibility of heterogeneous binding throughout the bead network depending on the resin saturation. By application of the broken rod model and under resin saturation it was proposed that an average of 1.2 antibodies adsorb per Protein A ligand in MabSelect SuRe at the outermost domains. Further investigation was performed at different surface concentrations in order to evaluate differences in the organization and stoichiometry in the different zones of the isotherm. The experimental data, analysed by the pearl necklace model, was compared with crystallographic structures of an IgG1 and a tetrameric chain of the B domain of Staphylococcal Protein A (the native form of the Protein A ligand present in MabSelect SuRe). It was found that at low isotherm concentrations the antibody to Protein A ratio was 1:1 and that at intermediate and high concentrations the 2:1 stoichiometry became favoured. The stoichiometry of 3:1 was also tested but was disregarded because of the strong steric effects. The offered approach in this thesis follows the adsorption process in situ, in the column and opens up new prospects to deeper investigation of all modes of chromatography of high industrial relevance, where the understanding of biomolecule–resin mechanism of interaction is of utmost importance.
O mercado de anticorpos monoclonais (mAbs – do inglês monoclonal antibodies) tem vindo a crescer exponencialmente ao longo das últimas décadas devido à elevada capacidade de resposta, selectividade, e robustez destas biomoléculas. O número de áreas de aplicação terapêutica dos mAbs tem também vindo a aumentar, sendo o cancro e as doenças autoimunes as mais representadas. Existem actualmente mais de 50 produtos aprovados e comercializados, representando uma receita de cerca de 100 mil milhões de dólares em vendas. Deste modo, devido à elevada procura e concomitante necessidade de aumento da produção destas biomoléculas, a indústria farmacêutica tem estado em constante evolução e optimização dos processos de produção e purificação de mAbs. Existem ainda critérios cada vez mais apertados para o controlo de qualidade destas biomoléculas por parte das principais agências reguladoras mundiais, nomeadamente a U.S. Food and Drug Administration (FDA) e a Agência Europeia do Medicamento (EMA – do inglês European Medicines Agency), de modo a assegurar a formulação de um produto seguro e de elevada pureza. É, por isso, necessário haver uma compreensão completa de todos os passos envolvidos em toda a cadeia de produção de anticorpos monoclonais. Um dos passos mais críticos, dispendiosos, e limitante é o passo de captura dos anticorpos durante a fase de purificação, nomeadamente o uso de cromatografia de afinidade com resinas de proteína A. A cromatografia de proteína A é o método mais aplicado para a purificação de anticorpos devido à sua elevada selectividade e também devido à sua robustez. As resinas de cromatografia utilizadas têm elevadas capacidades de ligação dinâmica, muito devido ao facto de os seus ligandos serem cadeias com múltiplos locais de ligação. Apesar da ligação dos anticorpos à proteína A ser amplamente conhecida, ainda não existe muita informação acerca do mecanismo através do qual a interação ocorre. Há certos aspectos como a estequiometria, a ligação preferencial, e a orientação tanto da cadeia de proteína A como do anticorpo que ainda não estão muito claros. Este conhecimento pode ser utilizado para optimizar a performance da captura de mAbs, quer seja através do melhoramento das resinas, quer seja através da minimização de custos devido a melhores previsões através do estabelecimento de modelos que incorporem estes parâmetros. Na cromatografia de proteínas são utilizados alguns sensores que têm por base fornecer informação acerca da concentração (UV), pureza (cromatografia de exclusão molecular em HPLC – SEC-HPLC do inglês), potência (ressonância de plasma de superfície – SPR do inglês), e estrutura (dicroísmo circular – CD do inglês e dispersão de luz por ângulo múltiplo – MALS do inglês). No entanto, todos estes sensores recolhem informação após as moléculas terem passado pela coluna de cromatografia operando online no sistema, ou então são usadas offine. Nenhum dos detectores fornece informação sobre a ligação anticorpo-proteína A realmente "in situ". Este trabalho de doutoramento teve como objectivo a compreensão da interacção entre anticorpos e resinas de proteína A com recurso a técnicas de operação in situ, de modo a poder estabelecer um modelo que consiga prever a adsorção de anticorpo, a sua organização estrutural aquando da ligação, a sua migração ao longo da coluna, a sua eluição e consequente pureza e potência. Para tal, foram usadas duas resinas de proteína A comerciais (MabSelect SuRe e TOYOPEARL AF-rProtein A HC) conhecidas pelas suas cadeiras com múltiplos domínios de ligação (4 e 6, respectivamente) e foi utilizado um mAb comercial (trastuzumab). A microcalorimetria de fluxo (FMC – do inglês) foi usada extensivamente para obter os parâmetros termodinâmicos associados à adsorção de anticorpos às resinas de proteína A. O microcalorímetro consiste numa coluna cilíndrica de 6 mm de diâmetro interno e 6 mm de altura com dois sensores térmicos acoplados às paredes da coluna capazes de detectar pequenas variações de potencial durante o processo cromatográfico. O perfil de adsorção mostrou ser de natureza exotérmica com dois passos subjacentes. Um primeiro momento é relativo à ligação em si, que resulta em grandes libertações de calor. Posteriormente, há uma reorganização dos anticorpos nos ligandos de modo a arranjarem a posição energeticamente mais favorável. De modo a caracterizar as alterações estruturais do complexo anticorpo-proteína A e avaliar a sua influência na topologia da superfície na adsorção, foi utilizada a técnica difracção de raios-X de pequeno ângulo (SAXS – do inglês small angle X-ray scattering). Foi usado um pequeno capilar de quartzo transparente aos raios-X em que foram empacotadas as resinas de proteína A. Foi possível acompanhar a formação da camada de anticorpo à superfície das resinas à medida que o anticorpo era introduzido. Foi demonstrada a possibilidade de ligação heterogénea dependendo da saturação da resina. Um modelo aplicado para interpretar os resultados foi o “broken rod model”, que sugere que as moléculas de anticorpo se ligam aos ligandos de proteína A no domínio mais exterior. Uma investigação posterior envolvendo diferentes concentrações de anticorpo foi realizada para avaliar a estequiometria de ligação em diferentes zonas da isotérmica. Os dados experimentais foram comparados com modelos cristalográficos reproduzindo o anticorpo e uma cadeia com quatro domínios de ligação semelhante à usada na resina MabSelect SuRe. Foi verificado que a baixas concentrações a estequiometria mais favorável é de 1:1 e que a concentrações intermédias 2:1 torna-se mais favorável, sendo sempre uma mistura de ambas. A estequimetria 3:1 foi igualmente testada e tida como possível, mas posteriormente desconsiderada como provável devido aos elevados efeitos estéricos presentes, pelo que esta condição carece de uma modelação mais avançada para poder ter em conta a flexibilidade das moléculas. Todos estes resultados confirmam a natureza heterogénea das resinas de proteína A. A abordagem oferecida por esta tese permitiu avaliar in situ a adsorção de anticorpos a proteína A durante o passo cromatográfico de afinidade, no entanto pode ser aplicada a qualquer tipo de cromatografia e para qualquer tipo de biomolécula, permitindo assim, abrir portas a uma investigação mais aprofundada para todos os tipos de cromatografia de alta relevância industrial, onde compreender o mecanismo de ligação biomolécula-ligando é de extrema importância.

碩士
明志科技大學
化學工程系生化工程碩士班
103
Polyacrylonitrile (PAN)nanofibrous membrane was prepared by electrospinning technique. The PAN membrane used in this work comprises a polyethylene terephthalate (PET) spunbond fabric as a supporting layer with upper and lower PANnanofibrous membrane. After 3M NaOH and diluted HCl treatments, the weak cationic exchange membrane (i.e., P-COOH) was obtained. The P-COOH membrane was then functionalized with Tris(hydroxymethyl)aminomethane (i.e., Tris). In this study, lysozyme was chosen as a model protein.The physical properties of these two nanofibrousmembranes were characterized in terms of fiber diameter, porosity and pore size, specific area of the surface, FTIR and SEM analysis. The adsorption experiments were carried out in a well-mixed system under the various operating conditions (e.g. modification pH, adsorption pH, and the molar ratio of reactants, P-COOH/Tris). According to the experimental results, the optimal synthesis of P-Trisnanofibrous membrane and adsorption conditions for lysozyme can be obtained. The dynamic adsorption characteristics of the P-COOH and P-Tris membranes for lysozyme using membrane chromatography were assessed by measurements of the breakthrough curves. The influences of operating conditions (e.g., adsorption pH, lysozyme concentration, no. of sheet membrane, and flow rate) on the adsorption performance of membrane were investigated in an AKTA prime chromatographic system (GE Healthcare). On the basis of the experiments, the adsorption capacity of P-Tris membrane for lysozyme was higher as compared to the P-COOH membrane. Moreover, the one-step elution scheme (0.8 M NaCl, pH 12) was chosen for eluting the adsorbed lysozyme. The lysozyme fraction eluted by 0.8 M NaCl for P-COOH and P-Tris membrane was recovered with a yield of 80.9% and 80.0%, respectively.

碩士
國立中興大學
化學工程學系
93
Abstract Recent advances in genomics and proteomics have led to a need for efficient methods for the purification of recombinant proteins. Among the many purification techniques, immobilized metal affinity chromatography（IMAC） exhibits unique characteristics for the one-step purification of poly(His)-tagged proteins and has thus become a routine purification technique in research and is gaining significance in industrial applications. To facilitate the application of IMAC in industrial processes, it is necessary to understand the complicate interactions between protein molecules and the adsorbents. In this study, a systematic study aiming at understanding the chromatographic behaviors of four poly(His)-tagged recombinant proteins, possessing different molecular weights and numbers of subunits, was carried out in both native and denaturing conditions. Four adsorption models, Langmuir, Scatchard, Temkin, and Langmuir-Freundlich, were used to evaluate the major adsorption parameters. It was confirmed in this study that the adsorption parameters were strongly dependent on the sizes and the numbers of subunits of the proteins. The results obtained in this study provide crucial information for elucidating the chromatographic behaviors of proteins and are instrumental in the simulation, scale-up, and operation of industrial chromatographic processes.

碩士
國立中興大學
化學工程學系所
106
Immobilized metal ion affinity chromatography (IMAC) is widely used for the purification of recombinant proteins containing poly(His) tag. The zinc finger region-methyltransferase fusion protein used in this study does not have a histidine tag, but its zinc-bonded coordination region is naturally attractive to divalent metals. So this property allows to do purification by IMAC. The researches of this laboratory before used the affinity adsorption of tandem Zn2+ and Ni2+ adsorbents to achieve the purification effect. This research continued to use the characteristics of immobilized metal ion affinity chromatography can replace the metal ion, then immobilized Cu2+, Ni2+, Co2+, Zn2+ to study equilibrium adsorption of the target protein with the zinc finger block. We also used Langmuir isotherm to simulate the maximum adsorption amount (qmax) and the dissociation constant (Kd). Compared with the equilibrium adsorption results of four kinds of metal ions affinity adsorption, whether the pre-equilibration was in the case of 10 mM imidazole or not, the BC61 zinc finger protein has the largest adsorption capacity by Cu2+ adsorbent, and the best affinity by Co2+ adsorbent.

碩士
明志科技大學
化學工程系生化工程碩士班
103
Polyacrylonitrile (PAN) nanofiber membrane was prepared by electrospinning technique. After heat treatment and alkaline hydrolysis, the ion exchange membrane (namely P-COOH) was grafted with ethylenediamine (EDA) and chitosan molecule, respectively. The obtained P-NH2 and P-Chitosan membranes were then covalently immobilized with Reactive Blue 49 dye, respectively to be used as dye ligand affinity membranes. The two dyed membranes were characterized in terms of fiber diameter, porosity, pore size, ligand density and binding capacities. The membrane was applied to evaluate the binding capacity of a model protein, lysozyme, under various operating parameters (e.g., adsorption pH, EDA, chitosan and dye concentrations, ionic strength, and temperature) were studied in batch modes. Under these circumstances, the optimal synthesis of dyed membrane and adsorption conditions for lysozyme could be obtained. The dynamic adsorption characteristics of these two dyed membranes for lysozyme using membrane chromatography were assessed by measurements of the breakthrough curves. The influences of operating conditions (e.g., adsorption pH, lysozyme concentration, and flow rate) on the adsorption performance of membrane were investigated in an ÄKTA prime prime chromatographic system (GE Healthcare). On the basis of the experiments, the adsorption capacity of P-NH2-dye membrane for lysozyme was higher as compared to the P-Chitosan-dye membrane. Moreover, the one-step elution scheme (1.0 M NaCl, pH 4) was chosen for eluting the adsorbed lysozyme. The lysozyme fraction eluted by 1.0 M NaCl for P-NH2-dye and P-Chitosan-dye membrane was recovered with a yield of 99% and 97%, respectively at a flow rate of 0.1 ml/min.

碩士
國立中興大學
化學工程學系所
100
In this study, a five-step approach was designed to prepare an immobilized metal-ion affinity membrane (IMAM) having a high capacity for protein adsorption. Polyacrylonitrile (PAN) membranes were selected as substrates due to their excellent thermal stability and solvent resistance. The -CN groups on PAN membranes were converted to -COOH groups by immersion into 3 N NaOH at 85 oC for 10 min, then 1 M HCl at 25 oC for 2 h. Next, the membranes were chemically modified by reacting with ethylenediamine (EDA) and ethylene glycol diglycidyl ether (EGDGE) to produce terminal epoxy groups. Iminodiacetic acid (IDA) was bound to the above modified PAN membranes (0.2 M or 1 M IDA and 1 M Na2CO3, pH 11) at 80 oC for 12 h and further chelated with copper ions (0.05 M or 0.5 M CuSO4) at 25 oC for 4 h. The optimal conditions were found to be 60 oC and 3 h for EDA reaction, as well as 60 oC and 4 h for EGDGE reaction. The resultant copper ion capacity was 250 umol/ml (1.5 umol/mg) under these optimized conditions, and the related lysozyme, BSA and GFP adsorption capacity were 510 ug/cm2, 69 ug/cm2 and 72 ug/cm2, respectively. By further increasing the IDA and CuSO4 concentrations to 1 M and 0.5 M, the lysozyme and BSA adsorption capacity were improved to 517 (ug/cm2) and 82 (ug/cm2), respectively. By calculation, the membrane surface area occupied by one BSA molecule is 36 nm2, close to its molecular size. However, the value for lysozyme is 0.95 nm2, indicating a jam-packed adsorption.

碩士
中興大學
化學工程學系所
95
The effects of molecular weight and number of poly(His)-tags on the adsorption of recombinant proteins on a hydroxyapatite-based immobilized metal affinity adsorbent were investigated in this study. Under native conditions, the adsorption isotherms were well-fitted by the Langmuir-Freundlich isotherm model. The maximum adsorption capacity was found to decrease with the increase in molecular weight. The binding affinity increased significantly with the number of poly(His)-tags. The adsorption capacities for esterase and epimerase were exceptionally high due to probably multilayer adsorption. Under denaturing condition, both the Langmuir and the Langmuir-Freundlich isotherm models were found to well-fitted the adsorption behavior of all five proteins studied. Under denaturing conditions, the binding affinities for all five proteins studied were essential identical and were much lower than that under native conditions, indicating that the Fe(III)-charged hydroxyapatite may not be appropriate for protein purification.

碩士
明志科技大學
生化工程研究所
102
In this work, polyacrylonitrile nanofiber membrane (PAN) was prepared by using electrostatic spinning technique. After a series of treatment for the PAN membrane, the modified membrane was finally functionalized with bromoacetic acidic (BrA), and iminodiacetic acidic (IDA) groups, respectively. The modified membranes wre chelated by Cu(II), Co(II), Ni(II), or Zn(II) as immobilized metal affinity nanofibrous (IMAN) membrane (abbrev. BrA-M or IDA-M). Recombinant E. coli cells containing enhanced green fluorescent protein (EGFP) was disrupted by ultrasonic technique. The isotherm and kinetic adsorption experiments were carried out by using clarified feedstock in a bath stirred tank system. The isotherm data for general protein and EGFP were well correlated by the Freundlich model for both IMAN membranes chelated by these metal ions. In kinetic experiments, the kinetic data for IMAN membrane were tested by using pseudo-first-order reaction and pseudo-second-order models. Generally speaking, the kinetic studies for general protein and EGFP showed that the adsorption followed a pseudo-second-order reaction. Its rate constant k2 increased with increasing concentrations of adsorption, indicating that the increased concentration of E. coli sample accelerated the adsorption rate. In membrane adsorption chromatography for both IMAN membranes, the dynamic binding capacity (DBC) was evaluated by adsorption breakthrough curve and calculated under different chelated metal ions and flow rates, respectively. The DBC for IMAN membrane was found to be lower at higher operating flow rate. For IDA-M adsorption process at a flow rate of 0.1 ml/min, the order of DBC for the general protein was Co(II) (40.0 mg/g)= Zn(II) (40.0 mg/g )> Cu(II) (16.2 mg/g)> Ni(II) (16.2 mg/g); the order of DBC for EGFP was Co(II) (5.77×106 AU/g)> Zn(II) (4.77×106 AU/g)> Cu(II) (3.47×106 AU/g)> Ni(II) (2.73×106 AU/g). However, for BrA-M adsorption process at the same flow rate, the order of DBC for the general protein was Ni(II) (31.6 mg/g)> Cu(II) (27.4 mg/g )> Zn(II) (21.4 mg/g)> Co(II) (10.6 mg/g); the order of DBC for EGFP was Ni(II) (8.25×106 AU/g)> Cu(II) (4.90×106 AU/g)> Co(II) (3.60×106 AU/g)> Zn(II) (3.41×106 AU/g). Based on the adsorption breakthrough curves, the results showed that IDA-M or BrA-M as IMAN membrane was not suitable for use in the purification of EGFP by using membrane chromatography.

碩士
國立中興大學
化學工程學系所
97
The adsorption behaviors of three recombinant proteins containing poly(histidine) tags on hydroxyapatite-based immobilized metal ion affinity chromatography (IMAC) are investigated in this study .The experimental data are well fitted with Langmuir–Freundlich isothermal adsorption model, indicating of positive cooperativity for the adsorption of these model proteins. The adsorption capacity and the binding affinity of the adsorbent for the model proteins are respectively dependent on the size and the number of poly(histidine) tags of proteins. The adsorption isotherms under denaturing conditions are well fitted with the Langmuir model and Langmuir–Freundlich model. The Scatchard analysis further suggest the homogeneous adsorption of the model protein subunits under denaturing conditions. The binding capacities and affinities under denaturing conditions for the three unfolded protein subunits become essentially identical because the molecular size and number of poly(His) tags of the unfolded polypeptide chains of the three protein subunits are the same.

碩士
國立成功大學
化學工程學系
89
Abstract This research is aimed at the use of chitosan as the supporting material of various adsorbents. CNBr activated and cross-linked method are used respectively to treat the chitosan for better adsorption results. Chitosan is mainly applied to the immobilized metal affinity chromatography in this work. -Amylase is the target adsorbate respectively in this study. The result shows that the best adsorption is formed by cross-linking chitosan with glutaraldehyde. In addition, when IDA or EDTA is used as the ligand, either one has very high chelating ability toward copper ion. Best adsorption for -amylase can only be found for cross-linked chitosan with Cu2+-chelated IDA (or EDTA) ligand. 0.2 M imidazole is determined to be a good and effective desorbent during desorption stage. For instance, by using imidazole (0.2 M) to desorb the -amylase from CNBr activated chitosan-IDA-Cu2+ can obtain 98% of the enzyme activity. In conclusion, this work applies immobilized affinity adsorbents for the adsorption of -amylase. Especially, chitosan is shown to be a good supporting material for such approach.

碩士
明志科技大學
化學工程系生化工程碩士班
105
In this study, the cellulose acetate (CA) nanofiber membrane was prepared by electrospinning technique. The operating parameters was to control the diameter including CA concentration, applied voltage, spinneret tip size and collector distance. The optimal conditions of electrospinning process for CA nanofiber membrane was found to be 25 kV (applied voltage), 12.5 mm (distance), and 0.016 mL/min (flow rate). The obtained CA nanofiber membrane was then treated with 0.1 M NaOH in H2O/ethanol (4:1) for 3-12 hr to obtain regenerated cellulose (RC) nanofiber membrane. The RC nanofiber membrane was further surface functionalized with reactive orange 4 dye, as a dye pseudo-affinity nanofiber membrane. To investigate the adsorption characteristics of dyed nanfiber membrane for malate dehydrogenase (MDH), some operating parameters were investigated, such as the hydrolysis time of RC nanofiber membrane, the immobilized density of dye, adsorption pH, and disrupted yeast concentration. The optimal dye immobilized density and adsorption pH were initial dye concentration of 5.0 mg/mL and pH value of 8.0, respectively. Finally, the elution efficiency for the adsorbed MDH by dyed membrane was investigated under various conditions, such as elution pH, salt concentration, additive reagent and its concentration. The preliminary results showed that the recovery and purification factor were found to be 51.39% and 30.10 folds, respectively with 1 M NaCl in 10% ethylene glycol at pH 5 acetate buffer. Hence, the dyed nanofiber member has potential for use in the purification of MDH from clarified yeast homogenate.

碩士
國立成功大學
化學工程學系碩博士班
90
Purification is essential in the biochemical industry. The approach of affinity chromatography is widely applied because of its specificity as well as its separate efficiency. The aim of this dissertation is to discuss the absorption and desorption the immobilized metal affinity adsorbent for alpha-amylase. In this work, the matrix beta-CD was cross-linked with EPI, then further immobilized with the ligand such as IDA, cibacron blue F3G-A, and DADPA respectively. In addition Cu2+ was chelated on the matrix to form immobilized metal affinity adsorbent. Among these adsorbents being studied, beta-CD CL-DADPA-Cu2+ shows best adsorption result of 99%. Meanwhile, of EDTA shows best result of desorption which can reach up to 98%. In conclusion, the affinity adsorbent beta-CD CL-DADPA-Cu2+ can reach best performance on adsorption and desorption.

Bentonite clays have long been used as additives in animal feed, aiming to improve pellet quality and prevent caking. Certain bentonites are also capable of deactivating aflatoxin B_(1) (AfB_(1)) in feed by adsorption, therefore, detoxifying the feed. However, a 10–fold difference in adsorption capacity has been observed among selected bentonites. The major mineralogical and chemical properties of smectites in determining their adsorption capacities for AfB_(1) are still poorly understood. Improved knowledge of the key controlling factors of aflatoxin adsorption to bentonite clays is needed to guide the selection, modification, and application of the clays as aflatoxin binders. The objective of this study was to test a hypothesis that a smectite's selectivity and adsorption capacity for aflatoxin was mainly determined by the size matching requirement on interlayer surface domains and the aflatoxin molecules. Three approaches were used to vary the size of nanometer-scaled nonpolar domains in the interlayer of smectites: 1) exchanging interlayer cations, 2) selecting natural bentonites with different cation exchange capacities (CEC), and 3) reducing charge density of a high CEC smectite. Six bentonites were fractionated, with their major mineralogical and chemical properties determined. Clay suspensions saturated with different cations were tested for aflatoxin adsorption. Some aflatoxin-smectite complexes were prepared and analyzed with FTIR and XRD. AfB_(1) adsorption isotherms were fitted with Langmuir, modified Langmuir with adsorption dependent affinity, and exponential Langmuir models. Divalent exchange cations with low hydration energy in general resulted in a much higher adsorption capacity and affinity for all six natural bentonite clays. Cations with smaller hydration radii tended to further enhance the adsorption process for aflatoxin on smectites. Charge density of smectite had shown significant effects on the adsorption capacity, affinity, and the isotherm shape. Aflatoxin adsorption isotherms on the six natural smectites and the CEC-reduced 5OK samples by Hofmann and Klemen effects suggested that there is an optimal CEC range between 80~110 cmol(+)/kg for the best aflatoxin binding smectites. When the smectite has a CEC within this range, the mineral has the highest affinity and adsorption capacity for AfB_(1). The aflatoxin adsorption results after cation exchange treatment, selection of different CEC smectites, and the CEC reduction on 5OK confirmed the importance of size and polarity matching on the nanometer scale in smectites’ adsorption for AfB_(1). All clay samples tested in this study were capable of adsorbing aflatoxin into interlayers, and the charge density seemed to have no effect on bonding strength.

碩士
國立臺灣海洋大學
食品科學系
92
DNA mismatches occur because of incorporation of noncomplementary, Watson-Crick bases opposite each other in the DNA helix during replication or recombination. Transition mispairs (G-T or A-C) are repaired by the mismatch repair process more efficiently than transversion mispairs (G-G, A-A, G-A, C-C, C-T, and T-T).In this study, a G-T probe was used to detect mismatch binding proteins. DNA mismatch binding activities in the extracts of the unicellular alga Chlorella pyrenoidosa were examined by electrophoretic mobility shift assay(EMSA). A protein concentration-dependent binding study revealed the presence of mismatch binding activities having low or no affinity for homoduplex DNA. Addition of 0.3 and 1.0 mM ATP to EMSA mixtures induced the formation of some high-shifting complexes reflecting the regulation of ATP on DNA mismatch recognition. Two polypeptides about 62 and 48 kDa estimated by SDS-PAGE were found to bind with high specificity to a biotin-labeled G-T probe immobilized on streptavidin-conjugated agarose beads and a few 62-kDa G-T binding polypeptides possessing pIs ranging from 5.4 to 5.8 were identified by two-dimensional gel electrophoresis after silver staining. Staining of affinity-captured proteins on a SDS-polyacrylamide gel by fluorescent SYPRO Ruby dye, however, detected a 13-kDa and a weak 48-kDa GT binding polypeptide. Two 13-kDa G-T binding polypeptides were found by fluorescence staining of 2-D gels. Peptide mass fingerprinting (PMF) of the 13-kDa polypeptide with pI5.3 matched best to a arabidopsis thaliana Serine/threonine protein phosphatase. The other polypeptide with pI5.5 matched best to a yeast ATP-dependent RNA helicase. The exact identities of the two polypeptides need to be detected.