Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins. Conventional purification processes are lengthy, technically complicated, product specific and time-consuming. To address this issue, herein we develop a one step purification system that due to the nature of the non-selective secretion system and the versatility of ion-exchange membrane chromatography can be widely applied to the production of many recombinant proteins. This was achieved through the integration of the intrinsically coupled upstream, midstream and downstream processes, a connection that is rarely exploited. A bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography medium while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange medium, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level of 487 U/L/OD600, with more than 90% was localized in the extracellular medium. Both, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. Furthermore, formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. iv At harvest, the whole non-clarified culture broth was applied directly to a tangential flow filtration anion-exchange membrane chromatography system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry). Due to their size, cells remained in the retentate while the extracellular medium penetrated the membrane. Most contaminate proteins were captured by the anion-exchange membrane, whereas the purified PAC was collected in the filtrate. The batch time for both cultivation and purification was less than 24 h and recombinant PAC with high purity (19 U/mg), process yield (74%), and productivity (41 mg/L) was obtained.

Polymeric nanoparticles are potential candidates as carriers for pharmaceutical agents. Development of such nanoparticles generally requires molecules immobilized on the particle surfaces to ensure biocompatibility and/or targeting abilities. Following particle preparation and surface modification, excess reagents must be removed. Ultracentrifugation, which is the most widely used purification technique as per today, is not feasible in industrial applications. In this diploma work, tangential flow filtration is studied as an alternative purification method which is better suited for implementation in a large-scale process. Comparison of ultracentrifugation and tangential flow filtration in diafiltration mode for purification of nanoparticles, indicate that they are comparable with respect to particle stability and the removal of the surfactant SDS from methacrylic anhydride nanoparticles. The purification efficiency of tangential flow filtration is superior to that of ultracentrifugation. Conductivity measurements of filtrates and supernatant liquids show that a stable conductivity value can be reached 6 times faster in filtration than in centrifugation with equipment and settings used. This conductivity arises from several types of molecules, and the contribution from surfactant molecules alone is not known. However, protein adsorption on the particles indicates successful removal of surfactant. Conductivity and tensiometry were evaluated as potential methods to quantify surfactant in solutions, but both proved unsatisfactory. Using bovine serum albumin as a model protein, the extent of immobilization to nanoparticles is evaluated at different pH. A maximum amount of 6,8 mg/m2 is immobilized, whereof an unknown part is covalently bound. This coverage is achieved at pH 4,0 and is probably partly due to low electrostatic repulsion between particle and protein. An estimation of 2,0 µmol covalently bound BSA per gram of nanoparticles corresponds to 5,3 mg/m2 and a surface coverage of 76%. Removal of excess reagents after surface modification is done with ultracentrifugation instead of filtration, as particle aggregates present after the immobilization reaction might foul the membrane.

<p>Polymeric nanoparticles are potential candidates as carriers for pharmaceutical agents. Development of such nanoparticles generally requires molecules immobilized on the particle surfaces to ensure biocompatibility and/or targeting abilities. Following particle preparation and surface modification, excess reagents must be removed. Ultracentrifugation, which is the most widely used purification technique as per today, is not feasible in industrial applications. In this diploma work, tangential flow filtration is studied as an alternative purification method which is better suited for implementation in a large-scale process.</p><p>Comparison of ultracentrifugation and tangential flow filtration in diafiltration mode for purification of nanoparticles, indicate that they are comparable with respect to particle stability and the removal of the surfactant SDS from methacrylic anhydride nanoparticles. The purification efficiency of tangential flow filtration is superior to that of ultracentrifugation. Conductivity measurements of filtrates and supernatant liquids show that a stable conductivity value can be reached 6 times faster in filtration than in centrifugation with equipment and settings used. This conductivity arises from several types of molecules, and the contribution from surfactant molecules alone is not known. However, protein adsorption on the particles indicates successful removal of surfactant. Conductivity and tensiometry were evaluated as potential methods to quantify surfactant in solutions, but both proved unsatisfactory.</p><p>Using bovine serum albumin as a model protein, the extent of immobilization to nanoparticles is evaluated at different pH. A maximum amount of 6,8 mg/m2 is immobilized, whereof an unknown part is covalently bound. This coverage is achieved at pH 4,0 and is probably partly due to low electrostatic repulsion between particle and protein. An estimation of 2,0 µmol covalently bound BSA per gram of nanoparticles corresponds to 5,3 mg/m2 and a surface coverage of 76%. Removal of excess reagents after surface modification is done with ultracentrifugation instead of filtration, as particle aggregates present after the immobilization reaction might foul the membrane.</p>

Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins. Conventional purification processes are lengthy, technically complicated, product specific and time-consuming. To address this issue, herein we develop a one step purification system that due to the nature of the non-selective secretion system and the versatility of ion-exchange membrane chromatography can be widely applied to the production of many recombinant proteins. This was achieved through the integration of the intrinsically coupled upstream, midstream and downstream processes, a connection that is rarely exploited. A bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography medium while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange medium, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level of 487 U/L/OD600, with more than 90% was localized in the extracellular medium. Both, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. Furthermore, formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. iv At harvest, the whole non-clarified culture broth was applied directly to a tangential flow filtration anion-exchange membrane chromatography system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry). Due to their size, cells remained in the retentate while the extracellular medium penetrated the membrane. Most contaminate proteins were captured by the anion-exchange membrane, whereas the purified PAC was collected in the filtrate. The batch time for both cultivation and purification was less than 24 h and recombinant PAC with high purity (19 U/mg), process yield (74%), and productivity (41 mg/L) was obtained.

Mikrosiebe stellen auf Grund ihrer Geometrie einen geringeren Strömungswiderstand im System dar und ermöglichen somit eine höhere Filtrationsgeschwindigkeit. Der Einsatz solcher Mikrosiebe anstelle herkömmlicher Membranen in Filtrationsprozessen kann deren Effizienz enorm steigern. Im Rahmen der Diplomarbeit wurden mittels partikelassistierter Benetzung hergestellte Mikrosiebe charakterisiert. Hierfür wurde zunächst ein Filtermodul für Kreuzstromfiltrationen entwickelt, welches anschließend in einen Versuchsaufbau integriert und charakterisiert wurde. Neben den Strömungswiderständen der rechteckigen Kanäle des Moduls wurde außerdem der Strömungswiderstand für Mikrosiebe einer Porengröße bestimmt und mit der Theorie verglichen. Es zeigt sich, dass Theorie und Praxis gut übereinstimmen und es sich bei dem entwickelten Versuchsaufbau um eine gute Methode handelt Strömungswiderstände von Mikrosieben zu bestimmen.

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O processo de microfiltração tangencial com membranas tubulares inorgânicas e tubos microporosos, respectivamente de origem importada e nacional, foi investigado tendo-se como objeto de separação as misturas macromoleculares preparadas com os agentes polissacarídeos gomas Xantana e Guar em suspensão aquosa. São evidenciados neste processo os efeitos de sinergia compreendidos nas misturas elaboradas puras e em diferentes proporções, na concentração final de 1000 ppm. A investigação experimental acompanha o comportamento reológico de cada mistura ao longo do processo e os resultados foram investigados a partir de propriedades físico-químicas relevantes como: concentração de carbono orgânico total (Total Organic Carbon - TOC) e análise de absorbância no espectômetro de infravermelho. Resultados de TOC indicaram que em apenas um meio filtrante tubular microporoso (C1T+), o desempenho do processo na retenção dos polissacarídeos foi acima de 90%, em relação ao processo com a membrana comercial importada cuja retenção de TOC foi da ordem de 80%. De acordo com as análises de absorbância, conclui-se que as membranas com tamanho do poro nominal de 0,2 \'mü\'m e 0,4 \'mü\'m tiveram uma retenção mais eficiente do que os tubos microporosos em estudo. A temperatura foi um parâmetro significativo, pois a retenção, na maioria dos casos, foi maior na temperatura de 25ºC, independente da pressão e da velocidade média. Na análise dos procedimentos envolvendo cada mistura, foi constatada a boa sinergia entre as gomas, sem modificação de estrutura (formação de gel) ou fenômenos de superfície que impedissem o processo de microfiltração. A vazão transmembrana das misturas ficou limitada entre os máximos valores para mistura pura de Xantana e mínimos para Guar. Todas as misturas apresentaram comportamento pseudoplástico, sem mudanças na reologia, reforçando a adequada sinergia quanto a este aspecto. / The present dissertation reports cross-flow microfiltration with national and imported inorganic tubular membranes and microporous tubes. The object of separation is the macromolecular mixtures prepared with polysaccharide agents Xanthan and Guar gum in aqueous solution. The process shows the synergism effects in pure and different mixture proportions, with 1000 ppm final concentration. The experiments established the rheologic behaviour of each mixture along the process and significant physical and chemical properties, such as Total Organic Carbon concentration (TOC) and absorbency analysis in infrared spectrometer were analyzed. TOC retention shows that only one microporous tubular filtering medium (C1T+) had better performance (retention 90%) than the imported commercial membrane (retention about 80%). Absorbency analysis show a better retention efficiency of membranes with pore size of 0,2 \'mü\'m and 0,4 \'mü\'m than microporous tube. The temperature was a significant parameter, as the better retention was obtained at 25ºC, independently of pressure and mean velocity. There is good synergism between Xanthan and Guar gums without structure modifications or surface phenomena that would impede the microfiltration process. The transmembrane flux of proportional mixtures has been limited between maximum values of pure Xanthan and minimum values of pure Guar mixtures. All mixtures have pseudoplastic behaviour, without changes in rheology, intensifying the good synergism.

O processo de microfiltração tangencial com membranas tubulares inorgânicas e tubos microporosos, respectivamente de origem importada e nacional, foi investigado tendo-se como objeto de separação as misturas macromoleculares preparadas com os agentes polissacarídeos gomas Xantana e Guar em suspensão aquosa. São evidenciados neste processo os efeitos de sinergia compreendidos nas misturas elaboradas puras e em diferentes proporções, na concentração final de 1000 ppm. A investigação experimental acompanha o comportamento reológico de cada mistura ao longo do processo e os resultados foram investigados a partir de propriedades físico-químicas relevantes como: concentração de carbono orgânico total (Total Organic Carbon - TOC) e análise de absorbância no espectômetro de infravermelho. Resultados de TOC indicaram que em apenas um meio filtrante tubular microporoso (C1T+), o desempenho do processo na retenção dos polissacarídeos foi acima de 90%, em relação ao processo com a membrana comercial importada cuja retenção de TOC foi da ordem de 80%. De acordo com as análises de absorbância, conclui-se que as membranas com tamanho do poro nominal de 0,2 \'mü\'m e 0,4 \'mü\'m tiveram uma retenção mais eficiente do que os tubos microporosos em estudo. A temperatura foi um parâmetro significativo, pois a retenção, na maioria dos casos, foi maior na temperatura de 25ºC, independente da pressão e da velocidade média. Na análise dos procedimentos envolvendo cada mistura, foi constatada a boa sinergia entre as gomas, sem modificação de estrutura (formação de gel) ou fenômenos de superfície que impedissem o processo de microfiltração. A vazão transmembrana das misturas ficou limitada entre os máximos valores para mistura pura de Xantana e mínimos para Guar. Todas as misturas apresentaram comportamento pseudoplástico, sem mudanças na reologia, reforçando a adequada sinergia quanto a este aspecto. / The present dissertation reports cross-flow microfiltration with national and imported inorganic tubular membranes and microporous tubes. The object of separation is the macromolecular mixtures prepared with polysaccharide agents Xanthan and Guar gum in aqueous solution. The process shows the synergism effects in pure and different mixture proportions, with 1000 ppm final concentration. The experiments established the rheologic behaviour of each mixture along the process and significant physical and chemical properties, such as Total Organic Carbon concentration (TOC) and absorbency analysis in infrared spectrometer were analyzed. TOC retention shows that only one microporous tubular filtering medium (C1T+) had better performance (retention 90%) than the imported commercial membrane (retention about 80%). Absorbency analysis show a better retention efficiency of membranes with pore size of 0,2 \'mü\'m and 0,4 \'mü\'m than microporous tube. The temperature was a significant parameter, as the better retention was obtained at 25ºC, independently of pressure and mean velocity. There is good synergism between Xanthan and Guar gums without structure modifications or surface phenomena that would impede the microfiltration process. The transmembrane flux of proportional mixtures has been limited between maximum values of pure Xanthan and minimum values of pure Guar mixtures. All mixtures have pseudoplastic behaviour, without changes in rheology, intensifying the good synergism.

Dans un contexte d’augmentation des volumes de boues résiduaires produits, comprendre et optimiser les processus de déshydratation mécanique est crucial. Leurs performances conditionnent tant le fonctionnement global de la station que son bilan environnemental et économique. En effet, la siccité des boues déshydratées impacte fortement les procédés ou filières permettant leur valorisation à l’échelle des territoires (compostage, valorisation agronomique directe, incinération, …) ainsi que leur bilan environnemental direct et indirect (tonnages à transporter, ...). De plus, les filtrats devront être traités par la station, et leur composition influence significativement le fonctionnement de la filière de traitement des eaux. Les filtres-presses font partie des procédés de déshydratation mécanique les plus utilisés dans les filières boues des stations d’épuration des eaux usées. Leurs performances sont fonction à la fois de leurs paramètres de fonctionnement et des propriétés de la boue à déshydrater. Les connaissances actuelles sont cependant insuffisantes pour permettre (1) d’expliquer pourquoi certaines boues se déshydratent mieux que d’autres et (2) de relier ces phénomènes aux processus mis en oeuvre lors de la déshydratation mécanique. Le poste de déshydratation fonctionne ainsi comme une boîte noire à partir de laquelle il est difficile de prédire les performances d’un outil de déshydratation appliqué à une boue particulière et d’anticiper des dysfonctionnements. Dans ce contexte, l’objectif de ce travail de thèse était de comprendre quels sont les facteurs de contrôle des performances de la déshydratation mécanique des boues d’épuration en filtre-presse et comment ces derniers impactent le fonctionnement du procédé. Dans un premier temps, l’impact des paramètres de fonctionnement des filtres-presses sur leurs performances a été étudié en cellule de filtration tangentielle (cellule de laboratoire reproduisant les processus d’un filtre presse industriel). Ce travail a permis de hiérarchiser les paramètres opératoires selon leur impact sur les performances de déshydratation. Cette étude a permis de mettre en évidence que le paramètre le plus sensible est l’épaisseur de gâteau suivi de la pression. L’impact de ces paramètres est en partie lié à celui du gradient de pression sur les équations de la filtration même si ce dernier doit être considéré en 3D pour expliquer les résultats observés. Dans un second temps l’étude de l’impact des propriétés des boues sur les performances de déshydratation a été réalisée. Pour ce faire, ce travail s’est focalisé sur l’impact d’un des procédés qui modifie le plus les propriétés des boues : la digestion anaérobie. L’objectif de ce travail était d’identifier comment le temps de séjour hydraulique (TSH) appliqué dans le digesteur influence les propriétés physicochimiques des boues et les performances de déshydratation évaluées au travers d’essais de déshydratation en cellule de filtration tangentielle. Cette phase a permis de montrer que quand le TSH augmente de 0 à un TSHcritique, la déshydratabilité de la boue diminue. Au-delà de ce TSHcritique, cette dernière s’améliore sans pour autant atteindre celle de la boue brute. Ces phénomènes ont pu être reliés à une modification des équilibres osmotiques entre les flocs et le milieu interstitiel couplée à une modification de la matrice d’exopolymères solubles des flocs. Enfin, un modèle numérique permettant de simuler la déshydratation des boues résiduaires a été implémenté sous COMSOL multiphysics®. Au travers des processus modélisés, il prend en compte les principaux facteurs de contrôle des performances identifiés auparavant. Après ajustement des paramètres sur un unique essai de déshydratation à une pression donnée, le modèle permet de prédire la siccité du gâteau pour une pression comprise entre 3 et 15 bars. / As more and more sewage sludge are generated, understanding and optimization of mechanical dewatering processes is crucial. Their performances affect both plant management and its environmental and economic impacts. Indeed, dryness of dewatered sludge strongly impacts processes for agronomic and energetic recovery (composting, direct agricultural recycling, incineration ...) and their direct and indirect environmental impacts (volumes to transport...). In addition, the filtrates have to be treated by the wastewater treatment plant, and their composition significantly affects its operating. Filter presses are one of the most common mechanical dewatering processes operated for sludge treatment in wastewater treatment plant. Their performance depends on their operating parameters and sludge properties. However, current knowledge is insufficient (1) to explain why some sludges are easier or harder to dewater than others, and (2) to link these phenomena to processes that carry out during the mechanical dewatering. Hence, dewatering works like a black box from which it is difficult to predict the efficiency of a dewatering tool performed with a particular sludge and to anticipate operating problems. In this context, the objective of this thesis was to understand witch factors drive the efficiency of mechanical dewatering of sewage sludge in the filter press and how they impact the process. First, the impact of operating parameters of filter presses on their efficiency was investigated in cross-flow filtration cell (lab-scale cross-flow filtration cell reproducing processes that occur in an industrial filter press). This work allowed the classification of the operating parameters according to their impact on the dewatering performances. This study highlighted that the most sensitive parameter is the cake thickness followed by pressure. The impact of these parameters is partially linked with their influence on the pressure gradient even if it should be considered in 3D to explain the results. Secondly, the study of the impact of sludge properties on dewatering performances was carried out. In this thesis, this work focuses on the impact of one of the processes which modifies most significantly sludge properties: anaerobic digestion. The objective of this work was to identify how hydraulic retention time (HRT) in the digester affects physicochemical properties of sludge and dewatering performance. Results demonstrated that when the HRT increases from 0 to a critical HRT (HRTcritical), sludge dewaterability decreases. Beyond this HRTcritical, it increases without reaching cake dryness of the raw sludge. These phenomena could be linked to a change in osmotic balance between the flocs and the bulk solution, combined with a modification of the soluble exopolymer matrix of flocs. Finally, a numerical model, allowing the simulation of sludge dewatering was implemented with COMSOL multiphysics®. Equation system takes into account the main factors that affect performances previously identified. After parameter calibration on a single dewatering experiment at 8 bars, the model allows the prediction of cake dryness for any pressure between 3 and 15 bars.