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Two transfected hybridoma cell lines TB/C3-bcl2, (overexpressing the Bcl-2 protein) and TB/C3-pEF (control cell line), were compared to assess the potential of Bcl-2 in preventing apoptosis. The conditions examined were batch cultures, growth in diluted media, and long-term fermentations in a high cell density fixed-bed reactor.

In this study the immortalised mouse hepatocyte line mHep-R1 was used for cultivation. The cells were first grown in culture flasks and in a small-scale fixed-bed system in order to determine growth characteristics and a suitable carrier type. The cell line was then cultivated in a 40 ml fixed-bed reactor over a period of 75 days at a perfusion rate of 6.25 ml medium per ml fixed-bed and day. A cell density of approx. 8.5·107 cells per ml carrier was reached at the end of the experiment proving that a stable cultivation was possible over a long period of time with constant consumption and production rates.

In this study the immortalised mouse hepatocyte line mHep-Rl was used for cultivation. The cells were first grown in culture flasks and in a small-scale fixed-bed system in order to determine growth characteristics and a suitable carrier type. The cell line was then cultivated in a 40 ml fixed-bed reactor over a period of 75 days at a perfusion rate of 6.25 ml medium per ml fixed-bed and day. A cell density of approx. 8.5.107 calls per ml carrier was reached at the end of the experiment proving that a stable cultivation was possible over a long period of time with constant consumption and production rates.

Nature has the impressive ability to efficiently and precisely control biological processes by applying highly evolved principles and using minimal space and relatively simple building blocks. The challenge is to transfer these principles into technically applicable and precise analytical systems that can be used for many applications. This article summarizes some of the new approaches in sensor technology and control strategies for different bioprocesses such as fermentations, biotransformations, and downstream processes. It focuses on bio- and chemosensors, optical sensors, DNA and protein chip technology, software sensors, and modern aspects of data evaluation for improved process monitoring and control.

An on-line feeding strategy based on oxygen uptake rate (OUR) was used to minimise the formation of inhibiting metabolites and to increase the yield of monoclonal antibodies in fed-batch cultures of hybridoma cells by a balanced supply of substrates. Concentrated feed was supplied according to the off-line estimated stoichiometric ratio between oxygen and glucose consumption (GC). The antibody concentration in fed-batch increased three-fold compared to the conventional batch culture by applying this strategy. Metabolites such as lactate and ammonia accumulated during fed-batch cultivation and it was not possible to avoid inhibition by ammonia. Fed-batch was efficiently improved by using the concept of dialysis and ‘nutrient-split’ feeding. where concentrated medium is fed directly io the cells and toxic metabolites are Temoved over a dialysis membrane into a buffer solution. This resulted in a ten-fold increase of the antibody concentration compared to the batch. Amino acid concentrations were analysed to identify limiting conditions during the cultivation and to analyse the performance of the nutrient supply in the fed-batch and dialysis fed-batch. The potential of the dialysis fed-batch with ‘nutrient-split’ feeding is discussed with respect to substrate loss, efficiency and process stability in comparison to the conventional fed-batch.

Pseudotype vectors are promising for gene transfer in many gene therapy approaches, however, low-vector concentration in batch cultures and high temperature-dependent decay do limit sufficiently large-scale production. To overcome these obstacles, the kinetic relations of cell growth and vector formation in different culture modes need to be understood. Effective optimisation of process modes is needed to achieve sufficient yields. Experimental and modelling studies were carried out in order to analyse the impact of different process modes such as perfusion, perfused fed-batch or repeated-batch on vector titer and productivity. Retroviral pseudotype vector, derived from the murine leukaemia virus carrying the HIV-1 envelop protein MLV (HIV-1) were produced using a 200 ml fixed bed reactor for high cell density cultivation on macroporous carriers. After starting the cultivation in batch mode, the reactor was either run in perfusion, perfused fed-batch or repeated-batch. A mathematical model of the bioreaction was developed on the basis of experimental data measured in culture dishes. The ability of the model to describe all different process modes of fixed-bed cultivation without additional fitting of the parameters was proven by three long-term cultivations for more than 400 h. The results of optimisation with the aid of the model, leads to the conclusion that perfusion with optimised harvest cycles and fed flows, result in a higher yield in comparison to batch or fed batch culture.

A flow-chamber bioreactor was designed for generation of three-dimensional cartilage-carrier-constructs. A specific attribute of the flow-chamber is a very thin medium layer for improved oxygen supply and a counter current flow of medium and gas. Three-dimensional cartilage-carrier-constructs were produced according to a standard protocol from chondrocytes of an adult mini-pig. The final step of this protocol was performed either in the bioreactor or in 12-well plates. The bioreactor experiments showed a significantly higher matrix thickness but a lower ratio of glycosaminoglycan to DNA. For both culture methods the constructs contained a high amount of collagen II. Appearance of the cartilage obtained in the bioreactor seemed to be closer to native cartilage with respect to distribution of the cells within the matrix, smoothness of the surface etc. All results considered the flow-chamber bioreactor is a very useful tool for generation of three dimensional cartilage-carrier constructs.

Three-dimensional cartilage-carrier-constructs were produced according to a standard protocol from chondrocytes of an adult mini-pig. Physical parameters (height and weight) correlated very well with total DNA content (r2 = 0.86, re. 0.94). The relation between DNA content and glycosaminoglycan content was less but still significant. No significant relationship was found between the elasticity module and the DNA content, even if the elasticity module increased slightly at higher DNA content. With respect to later implantation, selection of a construct for implantation based on the weight, which can be determined non-invasive and under sterile conditions, seems to be justifiable.

The determination of dissolved CO(2) and HCO(3)(-) concentrations as well as the carbon dioxide production rate in mammalian cell suspension culture is attracting more and more attention since the effects on major cell properties, such as cell growth rate, product quality/production rate, intracellular pH and apoptosis, have been revealed. But the determination of these parameters by gas analysis is complicated by the solution/dissolution of carbon dioxide in the culture medium. This means that the carbon dioxide transfer rate (CTR; which can easily be calculated from off-gas measurement) is not necessarily equal to carbon dioxide production rate (CPR). In this paper, a mathematical method to utilize off-gas measurement and culture pH for cell suspension culture is presented. The method takes pH changes, buffer and medium characteristics that effect CO(2) mass transfer into account. These calculations, based on a profound set of equations, allow the determination of the respiratory activity of the cells, as well as the determination of dissolved CO(2), HCO(3)(-) and total dissolved carbonate. The method is illustrated by application to experimental data. The calculated dissolved CO(2) concentrations are compared with measurements from an electrochemical CO(2) probe.

Background Advanced cell culture models, especially long-term 3D systems, require bioreactors allowing for cultivation under continuous flow conditions. Such culture models are for example tissue engineered implants, 3D cultures for drug testing, in vitro models of cell growth and migration for wound healing studies, cell cultures for biomaterial testing. New challenges in drug testing and biomaterial development arise from regulatory requirements. Animal trials have to be replaced by cell culture assays, preferably by test systems with human material. Standard 2D monolayer cultures are often unsatisfactory and therefore tissue-like 3D cultures are suggested as an alternative. Here the design of a multi-well flow-chamber bioreactor as a tool for manufacturing advanced cell culture models is presented. Advantages of this reactor concept can be seen in constant flow conditions, removal of toxic reaction products, high cell densities, and improved metabolism [1]. The general design of the flow chamber bioreactor (FCBR) can easily be modified for different applications and analytical requirements.

The supply of oxygen is a crucial parameter when cultivating animal cells in fixed-bed reactors because of the reaction-diffusion limitation within the porous carriers. To reduce limitation and increase productivity, the dissolved oxygen concentration was raised to above air saturation (hyperoxia) in long-term experiments using hybridoma cultures. This resulted in a threefold increase of the steady-state antibody production at high dilution rates compared to air saturated medium. A reaction-diffusion model was developed as a tool to describe the oxygen distribution in fixed-bed systems. The model corresponded well to the experimental data. It was also used to study the influence of several parameters on the performance of the fixed-bed system, such as the carrier size, the dissolved oxygen concentration, or the superficial flow velocity. By adapting the model it was shown that reaction-diffusion limitation is generally not a problem for other substrates such as glucose or glutamine.

A novel “nutrient-split” feeding strategy for cultivation of hybridoma and transfectoma cells was applied to a fixed bed reactor coupled with a dialysis membrane. Concentrated medium was fed directly to the fixed bed unit, whereas a buffer solution was used as dialysate. With both cell lines a steady state product concentration (monoclonal antibodies and Fab fragments, respectively) appr. 10 times higher compared to batch cultivation in T-flask was reached. A comparision with results of Bohmann et al. (1995), who employed the same experimental set-up and same hybridoma cell line, but used standard medium at both sides of the dialysis membrane, shows a much higher efficiency of the “nutrient-split” feeding strategy in respect to the extent of nutrient utilization.

A murine hybridoma cell line and a human/mouse transfectoma cell line were cultivated in three different media (serum-containing; low protein, serum-free; iron-rich, protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In the fixed bed reactor the cells were immobilized within macroporous carriers. Stirred suspension cultures of the hybridoma cells were only successfull in serum-containing medium. Using the serum-free media a lot of problems arose which are mainly due to the level of shear stress exposed to the cells. The transfectoma cell line did not grow in stirred suspension in any of the three media. For fixed bed cultivation of both cell lines, the serum-free media, which were optimized in flask cultures, could be used successfully as well as serum-containing medium.

In this work, we present a method to isolate mitochondria of mammalian cells after cell disruption on microscale. The device is composed of linear microchannels cast in PDMS (polydimethylsiloxane). Specific antibodies against the translocase outer membrane protein of the mitochondria are immobilized on the surface of the substrate using an avidin-biotin sandwich construct. The mitochondria can be captured in the channel, whereas the remains of the cell lysate flow out the chip unhindered. The captured mitochondria can be observed directly on chip. A successful immobilization of pre-isolated mitochondria was shown at a flow rate between 0 and 5 μl min(-1) (≈0-2.5 mm s(-1)). After fluorescence staining, we demonstrated that the mitochondria covered around 3% of the channel surface. The mitochondria appeared in a distinct spherical shape with a diameter of around 0.8-1.2 μm. Further validation of the microfluidic device using non-treated cell lysate was done at 2 μl min(-1). The immobilized mitochondria were smaller with a diameter of around ≈490 nm. We observed a surface coverage of around 4%. The immobilized mitochondria were active and stable for over 2 h without cooling and were shown to be able to produce ATP under stage 3 respiration on chip.

An industrial scale reactor concept for continuous cultivation of immobilized animal cells (e. g. hybridoma cells) in a radial-flow fixed bed is presented, where low molecular nutrients are supplied and low molecular weight metabolites are removed via dialysis membrane. High molecular products (e. g. monoclonal antibodies) are enriched. It will be demonstrated that lay-out for the large scale system can be done based on data determined in small scale.

The extremly thermophilic archaeon Pyrococcus furiosus was immobilized in macroporous AQUACEL carriers and cultivated continuously. Immobilized cell densities up to 4.6*10 9 cells ml - 1 related to the total medium volume were reached, corresponding to a cell density of 2.3*10 10 cells ml carrier - 1 . The cell activity could be maintained over long periods of time.

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran(®)-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.

The specific glucose uptake rate of a hybridoma cell line was determined in batch and continuous suspension cultures. For high density cultivation a membrane dialysis reactor was used. The aim was to obtain data, which can be used for process design and control. A decrease of the specific glucose uptake rate was observed during the course of batch cultures, even when the specific growth rate remained constant. These results are valid for cell densities between 2 x 10(5) and 2 x 10(6) ml-1. At cell densities between 2 x 10(6) and 1.5 x 10(7) ml-1 during continuous cultivation the specific glucose uptake rate remained constant. A relationship between the specific glucose uptake rate and the cell number was found and formulated in a mathematical equation. Literature data for different hybridoma cell lines fit in this plot very well. Applying this relationship, a correct estimation of the course of the glucose concentration in batch and continuous cultivation is possible.

Ammonia often has been reported to inhibit cell growth. The aqueous ammonia equilibrium between the un-ionized from (NH3) and the ammonium ion (NH4+) depends on the pH of the solution. Extensive studies in batch and continuous cultivation by varying pH and total ammonia concentration were carried out to investigate whether a kinetic model describing growth inhibition by ammonia has to be based on the total ammonia concentration, or the concentration of NH3. A significant relationship between the specific growth rate and death rate, respectively, and the NH3 concentration, but not the total ammonia concentration, was detected. An adaptation of the cells to high ammonia levels was not observed. Based on these results a new kinetic model for ammonia mediated growth inhibition is suggested. For high density cultivation it is recommended to control the pH at the lower limit of the growth optimum to keep the NH3 level low.

Extensive studies on the influence of the pH on the ammonia sensitivity of a murine hybridoma cell line were carried out in static flask cultures and at continuous cultivation in a stirred suspension reactor. A significant relationship between the specific growth rate and death rate, respectively, and the NH 3 concentration, but not the total ammonia concentration, was detected. Based on this results a new kinetic model for ammonia mediated growth inhibition is suggested.

For a hybridoma cell line a decrease of the glucose uptake rate was observed during the course of batch cultures, even when the growth rate remained constant. These results are valid for cell densities between 2*105 and 2*106 cells/ml. On the other hand these parameters remained constant at cell densities between 2*106 and 1.5*107 cells/ml during continuous cultivation. A relationship between the specific glucose uptake rate and the cell number could be found.

It could be shown that continuous cultivation of animal cells, e.g. hybridomas producing monoclonal antibodies (MAb’s) against penicillin-G-amidase, is possible in a membrane dialysis bioreactor with integrated radial-flow fixed bed over a period of 76 days. Because of the advantageous design of the reactor, the feed of serum has been proven to be necessary to the inner chamber only and therefore operation costs are reduced significantly compared to conventional reactor systems. Membrane fouling due to high protein titers was not observed. An operation mode of the reactor aiming at a high MAb productivity and concentration in the harvest stream was realized under the prerequisite of a constant glucose level. A mean maximum MAb concentration of 368 mg 1-1 was reached in continuous cultivation, ten times the value of stationary culture flasks after 4 days of incubation.

Stammzellbasierte zelltherapeutische Implantate: Entwicklung eines Herstellungsund Kryokonservierungsverfahrens Dipl.-Biol. Dipl.-Ing. (FH) C. Weber, S. Pohl, Prof. Dr.-Ing. R. Portner, Dr. C. Wallrapp, Dr. K. Hudel, N. Hofmann, Prof. Dr.-Ing. B. Glasmacher, Prof. Dr.-Ing. P. Czermak (E-Mail: peter.czermak@tg.fh-giessen.de) FH Giesen-Friedberg, Institut fur Biopharmazeutische Technologie, Wiesenstrase 14, D-35390 Giesen, Germany Technische Universitat Hamburg-Harburg, Bioprozessund Biosystemtechnik, Denickestrase 15, D-21071 Hamburg, Germany CellMed AG, Industriestrase 19, D-63755 Alzenau, Germany Martin Christ Gefriertrocknungsanlagen GmbH, An der Unteren Sose 50, D-37520 Osterode am Harz, Germany Leibniz Universitat Hannover, Institut fur Mehrphasenprozesse, Callinstrase 36, D-30167 Hannover, Germany Kansas State University, Dept. of Chemical Engineering, Durland Hall 105, Manhattan, KS 66506-5102, USA DOI: 10.1002/cite.201050007

Nowadays cell-based therapy is rarely in clinical practice because of the limited availability of appropriate cells. To apply cells therapeutically, they must not cause any immune response wherefore mainly autologous cells have been used up to now. The amount of vital cells in patients is limited, and under certain circumstances in highly degenerated tissues no vital cells are left. Moreover, the extraction of these cells is connected with additional surgery; also the expansion in vitro is difficult. Other approaches avoid these problems by using allo-or even xenogenic cells. These cells are more stable concerning their therapeutic behavior and can be produced in stock. To prevent an immune response caused by these cells, cell encapsulation (e.g. with alginate) can be performed. Certain studies showed that encapsulated allo- and xenogenic cells achieve promising results in treatment of several diseases. For such cell therapy approaches, stem cells, particularly mesenchymal stem cells, are an interesting cell source. This review deals on the one hand with the use of encapsulated cells, especially stem cells, in cell therapy and on the other hand with bioreactor systems for the expansion and differentiation of mesenchymal stem cells in reproducible and sufficient amounts for potential clinical use.

A hybridoma cell was cultivated continuously in a membrane dialysis bioreactor with an integrated radial-flow fixed bed consisting of porous Siran® carriers over a period of 6 weeks. Antibodies accumulated to an average of 100 mg l−1, approx. 10 times more than in fixed bed cultures without dialysis membrane. Serum costs could be reduced about 85% due to an appropriate feeding strategy. Siran® carriers with 3–5 mm diameter showed an advantage compared to those with 1–2 mm diameter. For the 3–5 mm carrier the specific glucose uptake rate and the MAb production rate were constant, if the velocity was between 0.09 mm s−1 and 0.75 mm s−1. At higher velocities cells are washed out of the bed. Furthermore antibody consistency and cell stability were verified in long-term cultivations over a period of 96 days. From an estimation of the antibody concentration reachable with the reactor concept under optimal conditions a concentration 45 times higher compared to axial-flow fixed bed reactors and 11 times higher compared to stirred tank reactors can be expected.

While fed-batch suspension culture of animal cells continues to be of industrial importance for the large-scale production of pharmaceutical products, existing control concepts are still insufficient. The present paper illustrates the advantages and disadvantages of different fed-batch strategies, including fixed-feed trajectories, control via OUR (oxygen uptake rate) (stoichiometric feeding), a priori determination of feed trajectories based on a kinetic model and the model-based adaptive OLFO (open-loop-feedback-optimal) control strategy. A recommendation as to which control strategy should be used for a specific process has to consider the respective process. For an established process with a well characterized and stable production cell line, probably the application of a fixed feed trajectory should be recommended. An adaptive, model-based control strategy could be the method of choice during cell-line development or for rapid production of small amounts of product for clinical trials, owing to its universal character and because it does not require intensive process development.

On etudie l'homogeneisation de solutions viscoelastiques et visqueuses de polymere dans un appareil agite et les caracteristiques de temps de melangeage

In fixed bed reactors with animal cells immobilized in macroporous carriers sufficient oxygen supply is a critical parameter. For modelling of the oxygen consumption and the oxygen profile in a fixed bed oxygen gradients within the porous carriers and along the length of the fixed bed have to be considered. For the complex geometry of the fixed bed a model structure was assumed, that allows the calculation of the oxygen profile. The model for oxygen supply of the immobilized cells included the transport resistance from the bulk fluid into the carriers and diffusion within the carriers.

Despite the advantages of mathematical bioprocess modeling, successful model implementation already starts with experimental planning and accordingly can fail at this early stage. For this study, two different modeling approaches (mechanistic and hybrid) based on a four-dimensional antibody-producing CHO fed-batch process are compared. Overall, 33 experiments are performed in the fractional factorial four-dimensional design space and separated into four different complex data partitions subsequently used for model comparison and evaluation. The mechanistic model demonstrates the advantage of prior knowledge (i.e., known equations) to get informative value relatively independently of the utilized data partition. The hybrid approach displayes a higher data dependency but simultaneously yielded a higher accuracy on all data partitions. Furthermore, our results demonstrate that independent of the chosen modeling framework, a smart selection of only four initial experiments can already yield a very good representation of a full design space independent of the chosen modeling structure. Academic and industry researchers are recommended to pay more attention to experimental planning to maximize the process understanding obtained from mathematical modeling. Bundesministerium für Bildung und Forschung (BMBF)

For the manufacturing of complex biopharmaceuticals using bioreactors with cultivated mammalian cells, high product concentration is an important objective. The phenotype of the cells in a reactor plays an important role. Are clonal cell populations showing high cell-specific growth rates more favorable than cell lines with higher cell-specific productivities or vice versa? Five clonal Chinese hamster ovary cell populations were analyzed based on the data of a 3-month-stability study. We adapted a mechanistic cell culture model to the experimental data of one such clonally derived cell population. Uncertainties and prior knowledge concerning model parameters were considered using Bayesian parameter estimations. This model was used then to define an inoculum train protocol. Based on this, we subsequently simulated the impacts of differences in growth rates (±10%) and production rates (±10% and ±50%) on the overall cultivation time, including making the inoculum train cultures; the final production phase, the volumetric titer in that bioreactor and the ratio of both, defined as overall process productivity. We showed thus unequivocally that growth rates have a higher impact (up to three times) on overall process productivity and for product output per year, whereas cells with higher productivity can potentially generate higher product concentrations in the production vessel.

Human mesenchymal stem cells (hMSC) are a promising cell source for the manufacturing of cell therapy or tissue-engineered implants. In part A of this publication a fixed-bed bioreactor system based on non-porous borosilicate glass spheres and procedures for the automated expansion of hMSC with high yield and vitality was introduced. Part B of this study deals with the modeling of the process in order to transfer the bioreactor system from the laboratory to the production scale. Relevant model parameters were obtained by fitting them to the experimental data of hMSC-TERT cultivations in scales up to 300 cm3. Scale-up calculations were carried out exemplarily for a target cell number of twenty billion cells.

For long term durability of tissue-engineered cartilage implanted in vivo, the development of the collagen fibre network orientation is essential as well as the distribution of collagen, since expanded chondrocytes are known to synthesise collagen type I. Typically, these properties differ strongly between native and tissue-engineered cartilage. Nonetheless, the clinical results of a pilot study with implanted tissue-engineered cartilage in pigs were surprisingly good. The purpose of this study was therefore to analyse if the structure and composition of the artificial cartilage tissue changes in the first 52 weeks after implantation. Thus, collagen network orientation and collagen type distribution in tissue-engineered cartilage-carrier-constructs implanted in the knee joints of Göttinger minipigs for 2, 26 or 52 weeks have been further investigated by processing digitised microscopy images of histological sections. The comparison to native cartilage demonstrated that fibre orientation over the cartilage depth has a clear tendency towards native cartilage with increasing time of implantation. After 2 weeks, the collagen fibres of the superficial zone were oriented parallel to the articular surface with little anisotropy present in the middle and deep zones. Overall, fibre orientation and collagen distribution within the implants were less homogenous than in native cartilage tissue. Despite a relatively low number of specimens, the consistent observation of a continuous approximation to native tissue is very promising and suggests that it may not be necessary to engineer the perfect tissue for implantation but rather to provide an intermediate solution to help the body to heal itself.

Human mesenchymal stem cells (hMSC) are a promising cell source for several applications of regenerative medicine. The cells employed are either autologous or allogenic; by using stem cell lines in particular, allogenic cells enable the production of therapeutic cell implants or tissue engineered implants in stock. For these purposes, the generally small initial cell number has to be increased; this requires the use of bioreactors, which offer controlled expansion of the hMSC under GMP-conform conditions. In this study, divided into part A and B, a fixed bed bioreactor system based on non-porous borosilicate glass spheres for the expansion of hMSC, demonstrated with the model cell line hMSC-TERT, is introduced. The system offers convenient automation of the inoculation, cultivation, and harvesting procedures. Furthermore, the bioreactor has a simple design which favors its manufacturing as a disposable unit. Part A is focused on the inoculation, cultivation, and harvesting procedures. Cultivations were performed in lab scales up to a bed volume of 300 cm3. The study showed that the fixed bed system, based on 2-mm borosilicate glass spheres, as well as the inoculation, cultivation, and harvesting procedures are suitable for the expansion of hMSC with high yield and vitality.

Determination par une methode photographique des temps de melangeage ou fonction du type d'agitateur utilise

L'etude (glucose, glucose-eau) experimentale menee avec des liquides newtoniens et non newtoniens (CMC) et un agitateur a disque permet la mise en place d'une correlation qui prend en compte la puissance et la vitesse de rotation de l'agitateur

Shake flask cultivation is nowadays a routine technique during process development for mammalian cell lines. During shaken culture, changes in agitation velocity, shaking diameter or shake flask size affect the hydrodynamics in the shake flask. This might be reflected in the growth of the cultured cells. Process parameters such as power input, mixing time, fluid velocity etc. have been determined and described mathematically for shake flasks used for microbial cultivation, but only to some extend for mammalian cell culture. Especially the relationship between these parameters and growth characteristics of mammalian cells is still a relatively uncovered issue. In this work, process parameters like specific power input, mixing time, maximum fluid velocity and Reynolds number were determined for four different shake flasks (baffled and unbaffled) in a range of shaking velocities on a shaking machine. The specific growth rate (μmax) of the human industrial cell line AGE1.HN® (ProBioGen AG, Berlin, Germany) was compared to the respective process parameters.

Background Experimental process-development and optimization is expensive and time-consuming. Real optimization by means of design of experiments involves data generation before optimization can be aimed for. This can make the way from process development to process establishment even harder, since academia or start-up research facilities might not have the possibility to generate these data. Furthermore, bioprocesses involving mammalian cells deal with many critical variables; processes are not only carried out batch wise, but increasingly in fedbatch mode with desired feeding profiles. The use of DoE tools in combination with an appropriate growth model might allow the experimenter to develop and to test fed-batch strategies in silico, before experiments are carried out in the laboratory. In our work, an unstructured model for mammalian cell culture was used for simulation. Kinetic parameters were derived from a small number of shake-flask experiments. The model was tested for data generation on common fed-batch strategies. By means of design of experiments strategies, relevant conditions were selected and experimentally tested. In this way, suitable fed-batch strategies for mammalian cell lines are evaluated in silico before bioreactor experiments are to be performed. This results in a significant reduction in the number of experiments during process development for mammalian cell culture.

Concept Production of biopharmaceuticals for diagnostic and therapeutic applications with suspension cells in bioreactors requires a seed train up to production scale [1]. For the final process steps in pilot and production scale the scale-up steps are usually defined (e.g. a factor of 5 10). More difficult in this respect are the first steps, the transitions between T-flasks, spinner tubes, roller bottles, shake flasks, stirred bioreactors or single-use reactors, because here often scale-up steps are different. The experimental effort to lay these steps out is correspondingly high. At the same time it is known that the first cultivation steps have a significant impact on the success or failure on production scale. The concept for a model based design of the seed train consists of the following steps