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See detailEtude de l’écoulement au sein d’un bioréacteur parallélépipédique à usage unique à agitation pendulo-elliptique
Collignon, Marie-Laure ULg; Droissart, Laurent; Delafosse, Angélique ULg et al

in Récents Progrès en Génie des procédés - A la croisée des sciences et des cultures, pour relever les défis industriels du XXIème siècle (2011, December 01)

Les bioréacteurs à usage unique sont de plus en plus envisagés dans l’industrie pharmaceutique en remplacement de leurs homologues en acier car ils permettent d’éliminer les étapes de lavage et de ... [more ▼]

Les bioréacteurs à usage unique sont de plus en plus envisagés dans l’industrie pharmaceutique en remplacement de leurs homologues en acier car ils permettent d’éliminer les étapes de lavage et de stérilisation tout en réduisant le risque de contamination croisée. Afin de s’insérer dans ce marché en expansion, les firmes ATMI LifeSciences, Pierre Guerrin et Artelis ont développé un bioréacteur à usage unique « Nucleo » composé d’une poche parallélépipédique en plastique souple mélangée par une pale réalisant un mouvement pendulo-elliptique. Différentes études ont démontré les bonnes performances de ce dispositif en termes d’homogénéisation et de maintien en suspension d’une phase solide ainsi que des productivités intéressantes lors de la réalisation de cultures de cellules animales. L’objectif de la présente étude est de caractériser l’écoulement du liquide au sein du bioréacteur grâce à des mesures réalisées par stéréo PIV. L’analyse des profils de vitesse a montré, qu’aux vitesses de rotation étudiées, l’écoulement au sein de la cuve est turbulent. Il apparaît que dans ces conditions, l’écoulement est complètement tridimensionnel et forme une spirale toroïdale plus ou moins déformée autour de l’axe central de la cuve. Il présente une certaine symétrie par rapport à cet axe central. Bien que la cuve soit parallélépipédique, aucune zone morte n’est observée dans les coins de celle-ci. [less ▲]

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See detailModèle hybride Euler-Lagrange pour la description des hétérogénéités dans les bioréacteurs.
Delafosse, Angélique ULg; Delvigne, Frank ULg; Collignon, Marie-Laure ULg et al

in SFGP (Ed.) Récents Progrès en Génie des Procédés - N° 101 - Des procédés au service du produit au coeur de l'Europe - Actes du XIIIème Congrès de la société Française de Génie des Procédés - Du 29 Nov. au 1er Décembre 2011 - Lille Grand Palais, FRANCE (2011, November 29)

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See detailPrise en compte l'état de la membrane cellulaire sur la réponse de biocapteurs fluorescents pour la détection de défaut d'écoulement au niveau des bioréacteurs : vers une intégration du "sécrétome" dans la problématique de l'extrapolation
Delvigne, Frank ULg; Brognaux, Alison ULg; Gorret, Nathalie et al

Conference (2011, November 29)

Malgré les nombreuses approches envisagées à ce jour, la dynamique du stress microbien en conditions de culture intensive (procédé fed-batch) est encore un aspect mal maîtrisé. Au cours de ce travail ... [more ▼]

Malgré les nombreuses approches envisagées à ce jour, la dynamique du stress microbien en conditions de culture intensive (procédé fed-batch) est encore un aspect mal maîtrisé. Au cours de ce travail, deux biocapteurs microbiens basés sur le principe de la protéine fluorescente verte (Green Fluorescent Protein ou GFP) ont été mis en œuvre dans des réacteurs scale-down permettant de reproduire les défauts d'écoulement rencontrés dans les bioréacteurs industriels. Les promoteurs de stress associés à la synthèse de la GFP ont été choisis suivant leur sensibilité à la limitation en source de carbone, condition standard qui est rencontrée dans les processus fed-batch ou l'apport en substrat carboné est limité afin d'éviter des déviations du métabolisme microbien (promoteurs rpoS et csiE). Les résultats obtenus montrent clairement que l'exposition des biocapteurs à des hétérogénéités locales de substrat entraîne une diminution de l'expression de la GFP. L'intensité de fluorescence a été mesurée à l'échelle cellulaire par cytométrie en flux. Durant les cultures, une chute significative du niveau de GFP intracellulaire a été observée pour les deux conditions scale-down considérées et pour les deux types de promoteur. Cette chute de fluorescence peut être attribuée à des phénomènes de répression des promoteurs suite à la levée locale de la limitation en carbone, mais également au relargage de la GFP dans le milieu extracellulaire. Ce relargage a été observé dans toutes les conditions opératoires considérées, comme le montrent les analyses des surnageants de culture par SDS-PAGE. L'intensité du relargage est néanmoins plus forte dans les conditions standard de culture (c'est-à-dire dans des réacteurs classiques sans approche scale-down). En effet, la coloration par l'iodure de propidium des biocapteurs cultivés en conditions standards est plus élevée que dans les conditions scale-down, suggérant une perméabilité membranaire plus élevée. Ces résultats offrent des potentialités intéressantes pour l'analyse simultanée de la viabilité cellulaire et de la détection des défauts d'écoulement par l'emploi de biocapteurs microbiens. De plus, les analyses par SDS-PAGE ont montre une grande diversité de protéines relargée en cours de procédé. Cette observation est étonnante du fait de l'emploi de milieux minéraux définis dans toutes les conditions étudiées. L'analyse de ce sécrétome offre des potentialités intéressantes pour la caractérisation des conditions de stress encourues par les micro-organismes au cours des procédés. [less ▲]

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See detailPotential use of GFP microbial biosensors for the detection of mixing imperfections and cell viability in bioreactors
Delvigne, Frank ULg; Delafosse, Angélique ULg; Collignon, Marie-Laure ULg et al

Conference (2011, September 25)

The dynamics of microbial stress response in intensive cultivation conditions remains misunderstood. In this work, two green fluorescent protein (GFP) transcriptional reporters have been used as ... [more ▼]

The dynamics of microbial stress response in intensive cultivation conditions remains misunderstood. In this work, two green fluorescent protein (GFP) transcriptional reporters have been used as biosensors of the heterogeneities generated in a two-compartment scale-down reactor. The stress promoters have been chosen for their responsiveness to carbon limitation corresponding to the global substrate profiles encountered in intensive fed-batch cultures. From our results, it can be concluded that the exposure of microbial cells to substrates heterogeneities tends to decrease the GFP expression level in fed-batch mode. Fluorescence intensities have been monitored at the single cell level by using flow cytometry. During the course of the fed-batch culture, a drop at the level of the intracellular GFP content has been observed for the two scale-down operating conditions and for the two promoters sensitive to substrate limitation (rpoS and csiE). The fluorescence drop can be attributed to the repression of these promoters but also to the release of GFP to the extracellular medium according to the increase of the fluorescence level of the supernatant. This leakage has been observed for all the operating conditions, i.e. the scale-down reactors and the culture operating in the normal mode. Interestingly, GFP leakage is more pronounced in the case of the cultures operated in the normal mode. Indeed, staining by propidium iodide (PI) tends to be more elevated for the microbial cells cultured under the normal mode by comparison with those cultured in scale-down conditions, indicating a higher permeability of the membrane. These results are in accordance with previously published ones (Hewitt and co-workers) suggesting that microbial cells cultivated in heterogeneous bioreactors (scale-down and large-scale bioreactors) exhibits a higher viability level. These results suggest that GFP microbial biosensors could be used to detect simultaneously mixing imperfections and their impact on the viability of microorganisms. [less ▲]

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See detailCharacterization of the response of GFP microbial biosensors sensitive to substrate limitation in scale-down bioreactors
Delvigne, Frank ULg; Brognaux, Alison ULg; Gorret, Nathalie et al

in Biochemical Engineering Journal (2011), 55(2), 131-139

The dynamics of microbial stress response in intensive cultivation conditions remains not completely understood. In this work, two green fluorescent protein (GFP) transcriptional reporters have been used ... [more ▼]

The dynamics of microbial stress response in intensive cultivation conditions remains not completely understood. In this work, two green fluorescent protein (GFP) transcriptional reporters have been used as biosensors of the heterogeneities generated in a two-compartment scale-down reactor. The stress promoters have been chosen for their responsiveness to carbon limitation corresponding to the global substrate profiles encountered in intensive fed-batch cultures. From our results, it can be concluded that the exposure of microbial cells to substrates heterogeneities tends to decrease the GFP expression level in fed-batch mode. Fluorescence intensities have been monitored at the single cell level by using flow cytometry. During the course of the fed-batch culture, a drop at the level of the intracellular GFP content has been observed for the two scale-down operating conditions and for the two promoters sensitive to substrate limitation (rpoS and csiE). The fluorescence drop can be attributed to the repression of these promoters but also to the release of GFP to the extracellular medium according to the increase of the fluorescence level of the supernatant. This leakage has been observed for all the operating conditions, i.e. the scale-down reactors and the culture operating in the normal mode, i.e. in a well-mixed bioreactor. Interestingly, GFP leakage is more pronounced in the case of the cultures operated in the normal mode. Indeed, staining by propidium iodide tends to be more elevated for the microbial cells cultured under the normal mode by comparison with those cultured in scale-down conditions, indicating a higher permeability of the membrane. These results suggest that GFP microbial biosensors could be used to detect simultaneously mixing imperfections and their impact on the viability of microorganisms. [less ▲]

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See detailReply to "Comments on Collignon et al. (Chem. Eng. Sci. 65 (2010) 5929-5941)"
Collignon, Marie-Laure ULg; Delafosse, Angélique ULg; Crine, Michel ULg et al

in Chemical Engineering Science (2011), 6

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See detailEstimation of the turbulent kinetic energy dissipation in a culture cell stirred tank bioreactor
Delafosse, Angélique ULg; Collignon, Marie-Laure ULg; Dossin, Denis et al

in Chemical Engineering Science (2011), 66(8), 1728-1737

The turbulent dissipation rate is a key parameter in stirred tanks and its local values may have a strong influence on the performance of many processes. However, the local dissipation rate estimation is ... [more ▼]

The turbulent dissipation rate is a key parameter in stirred tanks and its local values may have a strong influence on the performance of many processes. However, the local dissipation rate estimation is far from easy in a stirred tank, especially near the impeller discharge where maximum values are encountered. The aim of this work is to estimate the dissipation rate in a vessel used for animal-cell cultures and stirred with a down-pumping axial impeller (Mixel TTP) from velocity fields measured by 2D-PIV. Special attention is paid to the assumptions necessary to estimate the dissipation rate from 2D measurements and to the influence of measurement spatial resolution on the estimated values. The analysis of isotropy ratios measured on vertical, horizontal and tangential planes shows that the turbulence in the impeller discharge is far from isotropic. Isotropy assumptions classically used to estimate the dissipation rate from 2D measurements may thus lead to erroneous values. Based on the measured isotropy ratios, a new relationship is proposed to estimate the dissipation rate in the impeller discharge. This relationship is then used to estimate the dissipation rate on a vertical plane located in the impeller discharge zone. In order to analyze the influence of the measurement spatial resolution on the estimated values of the dissipation, a total of 12 spatial resolutions are tested. Results show that if the spatial resolution is divided by a factor 2, the dissipation rate increases by 220%. For the smallest spatial resolution value used, the maximum dissipation rate estimated is 50 times higher than the mean overall dissipation rate and the corresponding minimum value of the Kolmogorov scale is nearly 3 times smaller than the Kolmogorov scale computed from the mean overall dissipation rate. [less ▲]

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See detailAXIAL IMPELLER SELECTION FOR ANCHORAGE DEPENDENT ANIMAL CELL CULTURE IN STIRRED BIOREACTORS: METHODOLOGY BASED ON THE IMPELLER COMPARISON AT JUST-SUSPENDED SPEED OF ROTATION
Collignon, Marie-Laure ULg; Delafosse, Angélique ULg; Crine, Michel ULg et al

in Chemical Engineering Science (2010), 65(22), 5929-5941

Animal cells, which are nowadays essential for the industrial production of proteinic compounds, are commonly cultivated inside stirred tank bioreactors. In case of anchorage dependent cells, they are ... [more ▼]

Animal cells, which are nowadays essential for the industrial production of proteinic compounds, are commonly cultivated inside stirred tank bioreactors. In case of anchorage dependent cells, they are usually fixed on microcarriers. The choice of agitation conditions (impeller type, rotational speed…) in this type of process is not an easy task as it has to fulfil three potentially conflicting goals: (1) maintaining microcarriers in complete suspension, (2) homogenizing the culture medium, and (3) limiting mechanical constraints generated by the hydrodynamics on the cells. The aim of this study is to present an original methodology to select the most appropriate axial impeller for this specific application. Seven propellers are preselected on basis of their characteristics available in the literature. Instead of comparing impellers at a given rotational speed or a given power input, they are compared at their respective minimum impeller rotational speed that leads to a complete microcarrier suspension, i.e. at their respective just-suspended speed Njs. They are then compared at higher rotational speeds N, expressed as multiples of Njs. The impeller classification is based on the intensity of mechanical constraints they produced, evaluated from: (1) the macro-shear rate quantified by the spatial derivative of time average velocity fields measured by P.I.V, (2) the micro-shear rate characterized by the ratio between the microcarrier diameter to the average Kolmogorov scale computed from power input measurements, and (3) the impact of microcarrier collisions on cells described via the Turbulent Collision Severity index also computed from power input measurements. Results show that the 125 mm diameter TTP impeller (Mixel) and the 150 mm diameter Elephant Ear impeller (Applikon) produce the smallest mechanical constraints at their just-suspended speed (50 rpm and 20 rpm, respectively). Moreover, the mechanical constraints they produce increase more slowly with the N/Njs ratio than the mechanical constraints produced by other impellers. These propellers are thus even more advantageous if rotational speeds higher than the just-suspended speed have to be used. [less ▲]

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See detailDevelopment of a compartment model based on CFD simulations for mixing description in bioreactors
Delafosse, Angélique ULg; Delvigne, Frank ULg; Collignon, Marie-Laure ULg et al

in Biotechnologie, Agronomie, Société et Environnement = Biotechnology, Agronomy, Society and Environment [=BASE] (2010), 14(2), 517-522

Understanding and modelling the complex interactions between biological reaction and hydrodynamics is a key problem when dealing with bioprocesses. It is fundamental to be able to accurately predict the ... [more ▼]

Understanding and modelling the complex interactions between biological reaction and hydrodynamics is a key problem when dealing with bioprocesses. It is fundamental to be able to accurately predict the hydrodynamics behaviour of bioreactors of different size and its interaction with the biological reaction. CFD can provide detailed modelling about hydrodynamics and mixing. However, it is computationally intensive, especially when reactions are taken into account. Another way to predict hydrodynamics is the use of "Compartment" or "Multi-zone" model which are much less demanding in computation time than CFD. However, compartments and fluxes between them are often defined by considering global quantities not representative of the flow. To overcome the limitations of these two methods, a solution is to combine compartment modelling and CFD simulations. Therefore, the aim of this study is to develop a methodology in order to propose a compartment model based on CFD simulations of a bioreactor. The flow rate between two compartments can be easily computed from the velocity fields obtained by CFD. The difficulty lies in the definition of the zones in such a way they can be considered as perfectly mixed. The creation of the model compartments from CFD cells can be achieved manually or automatically. The manual zoning consists in aggregating CFD cells according to the user's wish. The automatic zoning defines compartments as regions within which the value of one or several properties are uniform with respect to a given tolerance. Both manual and automatic zoning methods have been developed and compared by simulating the mixing of an inert scalar. For the automatic zoning, several algorithms and different flow properties have been tested as criteria for the compartment creation. [less ▲]

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See detailQuality of mixing in a stirred bioreactor used for animal cells culture: heterogeneities in a lab scale bioreactor and evolution of mixing time with scale up
Collignon, Marie-Laure ULg; Dossin, Denis; Delafosse, Angélique ULg et al

in Biotechnologie, Agronomie, Société et Environnement = Biotechnology, Agronomy, Society and Environment [=BASE] (2010), 14

Animal cells are industrially cultivated inside stirred bioreactors to produce proteinic compounds. Due to the use of mild agitation conditions in order to limit mechanical constraints, the homogeneity of ... [more ▼]

Animal cells are industrially cultivated inside stirred bioreactors to produce proteinic compounds. Due to the use of mild agitation conditions in order to limit mechanical constraints, the homogeneity of the culture medium can be far from perfect. This study has therefore two objectives. 1) the global characterization of the mixing via the mixing time 2) the local description of concentration fields. The mixing time is measured by conductimetry inside 20 L, 80 L, 600L tanks. The Grenville correlation is adjusted on these experimental measurements to improve the prediction of the mixing time during the scale-up of the process. The concentration fields are visualized by the Planar Laser Induced Fluorescence (P.L.I.F) technique in the 20 L tank. This part of the study is focused on the time evolution of the maximum value of the tracer concentration inside measurement planes and of the numerical distribution of theses concentration fields. [less ▲]

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