Silicone-tubing aerated bioreactors for somatic embryo production

Bioreactors equipped with silicone tubings for bubble free oxygen supply are suitable for culture of embryogenic cell suspensions. The advantages of bubble free aeration systems over various devices for dispersion of air bubbles are the lack of foam formation and the possibilities of precise control of the desired oxygen set point. The specification of silicone tubing (length, diameter, wall thickness) has to be adapted according to the amount of embryogenic biomass to be produced in the bioreactor. Cell suspensions of Euphorbia pulcherrima were cultured --2 l bioreactor at 60% pO₂, supplied by a silicone tubing system of 155 cm length, 4.0 mm diameter and 0.4 mm wall thickness. The oxygen concentration decreased when the packed cell volume exceeded 14% (=3.7 g l^-1 cell dry weight), indicating the upper limit of oxygen supply by the silicone tubing. Mathematical considerations for membrane aerated bioreactors are presented with the intention of enabling a more precise definition for the configuration of silicone tube systems in different bioreactor types.     

Scaling-up vaccine production: implementation aspects of a biomass growth observer and controller

This study considers two aspects of the implementation of a biomass growth observer and specific growth rate controller in scale-up from small- to pilot-scale bioreactors towards a feasible bulk production process for whole-cell vaccine against whooping cough. The first is the calculation of the oxygen uptake rate, the starting point for online monitoring and control of biomass growth, taking into account the dynamics in the gas-phase. Mixing effects and delays are caused by amongst others the headspace and tubing to the analyzer. These gas phase dynamics are modelled using knowledge of the system in order to reconstruct oxygen consumption. The second aspect is to evaluate performance of the monitoring and control system with the required modifications of the oxygen consumption calculation on pilot-scale. In pilot-scale fed-batch cultivation good monitoring and control performance is obtained enabling a doubled concentration of bulk vaccine compared to standard batch production.     

Stepwise formation of patterned cell co-cultures in silicone tubing

Here, we describe a method for producing patterned cell adhesion inside silicone tubing. A platinum (Pt) needle microelectrode was inserted through the wall of the tubing and an oxidizing agent electrochemically generated at the inserted electrode. This agent caused local detachment of the anti-biofouling heparin layer from the inner surface of the tubing. The cell-adhesive protein fibronectin selectively adsorbed onto the newly exposed surface, making it possible to initiate a localized cell culture. The electrode could be readily set in place without breaking the tubular structure and, importantly, almost no culture solution leaked from the electrode insertion site after the electrode was removed. Ionic adsorption of poly-L-lysine at the tubular region retaining a heparin coating was used to switch the heparin surface from cell-repellent to cell-adhesive, thereby facilitating the adhesion of a second cell type. The combination of the electrode-based technique with layer-by-layer deposition enabled the formation of patterned co-cultures within the semi-closed tubular structure. The utility of this approach was demonstrated by patterning co-cultures of hepatocytes or endothelial cells with fibroblasts. The controlled co-cultures inside the elastic tubing should be of value for cell-cell interaction studies following application of chemical or mechanical stimuli and for tissue engineering-based bioreactors.     

Development of a single-pass ceramic matrix bioreactor for large-scale mammalian cell culture

A single-pass, plug-flow bioreactor has been developed in which oxygen is supplied to entrapped hybridoma cells via sllicone tubes threaded through the square channels of a macroporous ceramic monolith. Oxygen diffuses from the gas phase, through the silicone tubing, across the open square channel, and into the pores of the ceramic wall where it is consumed by entrapped cells. Advantages of such a reactor include higher product yields, protection of cells from detrimental hydrodynamic effects, no internal moving parts to compromise asepsis, and simplicity of operation. A prototype bioreactor was constructed and operated over a range of residence times. A side-by-side experimental comparison with a conventional recycle bioreactor was performed by inoculating both bioreactors with cells from the same stock culture and feeding medium from the same reservoir. Final antibody titers were 80% higher in the single-pass bioreactor at a residence time of 200 minutes compared with those of the recycle bioreactor at a residence time of 800 minutes. A theoretical analysis of oxygen transport in this bioreactor is developed to highlight important design criteria and operating strategies for scale-up. (c) 1992 John Wiley & Sons, Inc.     

Dynamic modeling and simulation of continuous airlift bioreactors

For dynamic behaviors of continuous airlift bioreactors, a mathematical model based on a tanks-in-series model with backflow has been developed. The equations describing the dynamics of airlift bioreactors are material balances for micro-organism, substrate, dissolved oxygen and oxygen in gas-phase and heat balances. Non-ideal mixing of liquid and gas phases is taken into account using a tanks-in-series model with backflow. The batch operation, startup operation and the consequence of plant failure were simulated and the effects of design and operating parameters for an airlift bioreactor on its dynamic behaviors were discussed. The concentration profiles of micro-organism, substrate, dissolved oxygen and oxygen in gas-phase and the temperature profile in an airlift bioreactors and their dynamics were obtained. The computational results indicate that the transients of a chemostat in the case of bubble column bioreactor are slower compared with those in the case of airlift bioreactor. The proposed simulator is more precise as compared with models published previously in the literature and therefore provides more reliable and rational examination of continuous airlift bioreactor performance.     

Advanced microscale bioreactor system: a representative scale-down model for bench-top bioreactors

In recent years, several automated scale-down bioreactor systems have been developed to increase efficiency in cell culture process development. ambr™ is an automated workstation that provides individual monitoring and control of culture dissolved oxygen and pH in single-use, stirred-tank bioreactors at a working volume of 10–15 mL. To evaluate the ambr™ system, we compared the performance of four recombinant Chinese hamster ovary cell lines in a fed-batch process in parallel ambr™, 2-L bench-top bioreactors, and shake flasks. Cultures in ambr™ matched 2-L bioreactors in controlling the environment (temperature, dissolved oxygen, and pH) and in culture performance (growth, viability, glucose, lactate, Na⁺, osmolality, titer, and product quality). However, cultures in shake flasks did not show comparable performance to the ambr™ and 2-L bioreactors.     

Antibody production by a hybridoma cell line at high cell density is limited by two independent mechanisms

Our previous attempt to model the stationary phase of production-scale hollow-fiber bioreactors using a scaled-down micro hollow-fiber bioreactor resulted in a predicted antibody production rate that was three- to fourfold lower than the actual value (Gramer and Poeschl, 2000). Medium limitations were suspected as the reason for the discrepancy. In this study, various increases in medium feed rate were implemented in the micro bioreactor by increasing the diameter of the silicone tubing that houses the hollow fibers. Because larger diameter tubing may induce oxygen limitations, we also explored the effect of medium recirculation to enhance oxygenation. Antibody production in the micro bioreactor increased both as a result of increased medium supply and due to medium recirculation. However, these parameters increased antibody production through two independent mechanisms. The increased medium supply resulted in a higher cell-specific antibody production rate, but not a higher viable cell density. Medium circulation resulted in the support of a higher viable cell density, but had little effect on the cell-specific secretion rate. The two mechanisms of enhanced antibody production were additive, demonstrating that simultaneous parameters can limit antibody production by this cell line in a hollow-fiber system. When the medium feed and circulation rates were increased to a volumetrically proportional scale, scale-up predictions from the micro bioreactor matched the actual data from the production-scale system to within 15%. These data demonstrate the usefulness of the micro bioreactor for characterizing cell growth and limiting mechanisms at high cell densities.     

Scale‐up analysis for a CHO cell culture process in large‐scale bioreactors

Bioprocess scale‐up is a fundamental component of process development in the biotechnology industry. When scaling up a mammalian cell culture process, it is important to consider factors such as mixing time, oxygen transfer, and carbon dioxide removal. In this study, cell‐free mixing studies were performed in production scale 5,000‐L bioreactors to evaluate scale‐up issues. Using the current bioreactor configuration, the 5,000‐L bioreactor had a lower oxygen transfer coefficient, longer mixing time, and lower carbon dioxide removal rate than that was observed in bench scale 5‐ and 20‐L bioreactors. The oxygen transfer threshold analysis indicates that the current 5,000‐L configuration can only support a maximum viable cell density of 7 × 10⁶ cells mL^?1. Moreover, experiments using a dual probe technique demonstrated that pH and dissolved oxygen gradients may exist in 5,000‐L bioreactors using the current configuration. Empirical equations were developed to predict mixing time, oxygen transfer coefficient, and carbon dioxide removal rate under different mixing‐related engineering parameters in the 5,000‐L bioreactors. These equations indicate that increasing bottom air sparging rate is more efficient than increasing power input in improving oxygen transfer and carbon dioxide removal. Furthermore, as the liquid volume increases in a production bioreactor operated in fed‐batch mode, bulk mixing becomes a challenge. The mixing studies suggest that the engineering parameters related to bulk mixing and carbon dioxide removal in the 5,000‐L bioreactors may need optimizing to mitigate the risk of different performance upon process scale‐up. Biotechnol. Bioeng. 2009;103: 733–746. © 2009 Wiley Periodicals, Inc.     

Engineering challenges in high density cell culture systems

High density cell culture systems offer the advantage of production of bio-pharmaceuticals in compact bioreactors with high volumetric production rates; however, these systems are difficult to design and operate. First of all, the cells have to be retained in the bioreactor by physical means during perfusion. The design of the cell retention is the key to performance of high density cell culture systems. Oxygenation and media design are also important for maximizing the cell number. In high density perfusion reactors, variable cell density, and hence the metabolic demand, require constant adjustment of perfusion rates. The use of cell specific perfusion rate (CSPR) control provides a constant environment to the cells resulting in consistent production. On-line measurement of cell density and metabolic activities can be used for the estimation of cell densities and the control of CSPR. Issues related to mass transfer and mixing become more important at high cell densities. Due to the difference in mass transfer coefficients for oxygen and CO₂, a significant accumulation of dissolved CO₂ is experienced with silicone tubing aeration. Also, mixing is observed to decrease at high densities. Base addition, if not properly done, could result in localized cell lysis and poor culture performance. Non-uniform mixing in reactors promotes the heterogeneity of the culture. Cell aggregation results in segregation of the cells within different mixing zones. This paper discusses these issues and makes recommendations for further development of high density cell culture bioreactors.     

Enhancing oxygen transfer in surface-aerated bioreactors by stable foams.

To enhance oxygen transfer in surface-aeration bioreactors, stabilized foams were generated to increase the gas-liquid interfacial area by slowly introducing coarse bubbles into media containing fetal bovine serum. The bubble sparging rates were so low (i.e., 20 and 50 mL/h) that the contribution to oxygen transfer from these bubbles was due to foaming instead of bubbling. Furthermore, no physical cell damage caused by bubble sparging was observed. Oxygen transfer coefficients, kLa, in the bioreactors were measured in cell-free media. Without the foam-stabilizing agent (i.e., serum), no appreciable change in kLa was observed due to the bubble sparging. On the other hand, with serum, kLa increased with increasing serum content and bubble sparging rate and corresponded well with the degree of foaming. With 10% fetal bovine serum and a bubble sparging rate of 50 mL/h, kLa increased approximately 90% compared with no foaming. The enhancing effect of foam on oxygen transfer in surface aeration bioreactors has been further demonstrated with hybridoma cultures simultaneously grown in three identical bioreactors with and without stabilized foams.     

Description and evaluation of reciprocating plate bioreactors

The environment in which live microorganisms has a major impact on their productivity. One important factor is the mechanical mixing that is used to promote good heat and mass transfer in bioreactors. In this paper, the performance of reciprocating plate bioreactors is first evaluated for their ability to produce high oxygen transfer coefficients. Pure water and a glycerol water (5050 wt%) solution are used for this evaluation. Then, the performance of reciprocating plate bioreactors for the production of an exocellular polysaccharide (pullulan) by yeast Aureobasidium pullulans is analyzed in terms of quantity and quality of the polysaccharide. Results clearly show that a more efficient substrate utilisation is achieved with reciprocating plate bioreactors.     

Device for sterile online measurement of the oxygen transfer rate in shaking flasks

The oxygen transfer rate (OTR) is the most suitable measurable parameter to quantify the physiological state of a culture of aerobic microorganisms since most metabolic activities depend on oxygen consumption. Online measurement of the oxygen transfer rate in stirred bioreactors is state of the art although technically difficult. However, the online determination of the oxygen transfer rate in shaking bioreactors under sterile conditions has not been possible until recently. A newly developed measuring device eliminates this deficit. Extremely useful information about cultivating conditions and the physiological state of microorganisms can be gained in early stages of research and bioprocess development from many reactors operated in parallel.     

The application of membrane biological reactors for the treatment of wastewaters

Combining membrane technology with biological reactors for the treatment of municipal and industrial wastewaters has led to the development of three generic membrane processes within bioreactors: for separation and recycle of solids; for bubbleless aeration of the bioreactor; and for extraction of priority organic pollutants from hostile industrial wastewaters. Commercial aerobic and anaerobic membrane separation bioreactors already provide a small footprint alternative to conventional biological treatment methods, producing a high-quality effluent at high organic loading rates. Both the bubbleless aeration and extractive membrane bioreactors are in the development stages. The former uses gas-permeable membranes to improve the mass transfer of oxygen to the bioreactor by providing bubbleless oxygen. By using a silicone membrane process, extractive membrane bioreactors transfer organic pollutants from chemically hostile wastewaters to a nutrient medium for subsequent biodegradation. All three membrane bioreactor (MBR) processes are comparatively and critically reviewed. (c) 1996 John Wiley & Sons, Inc.     

Controlled supply of trace amounts of oxygen in laboratory scale fermentations

A detailed study of anaerobic yeast fermentations requires controlled addition of minor amounts of oxygen to the fermentor. A method for supplying small-scale fermentations with millimolar amounts of oxygen is described. The controlled addition is accomplished by diffusion of oxygen through silicone tubing carrying nitrogen to the fermentor. Sulfite values of the fermentation system are given for different gas velocities and lengths of silicone tubing. Two experiments are outlined in which this technique is applied and the possible energy yield from oxydative phosphorylation is discussed.     

Methods and milliliter scale devices for high-throughput bioprocess design

Based on electromagnetic simulations as well as on computational fluid dynamics simulations gas-inducing impellers and their magnetic inductive drive were optimized for stirred-tank reactors on a 10 ml-scale arranged in a bioreaction block with 48 bioreactors. High impeller speeds of up to 4,000 rpm were achieved at very small electrical power inputs (63 W with 48 bioreactors). The maxima of local energy dissipation in the reaction medium were estimated to be up to 50 W L⁻¹ at 2,800 rpm. Total power input and local energy dissipation are thus well comparable to standard stirred-tank bioreactors. A prototype fluorescence reader for 8 bioreactors with immobilized fluorometric sensor spots was applied for online measurement of dissolved oxygen concentration making use of the phase detection method. A self-optimizing scheduling software was developed for parallel control of 48 bioreactors with a liquid-handling system for automation of titration and sampling. It was shown on the examples of simple parallel batch cultivations of Escherichia coli with different media compositions that high cell densities of up to 16.5 g L⁻¹ dry cell mass can be achieved without pH-control within 5 h with a high parallel reproducibility (standard deviation<3.5%, n=48) due to the high oxygen transfer capability of the gas-inducing stirred-tank bioreactors.     

Comparisons of the glycosylation of a monoclonal antibody produced under nominally identical cell culture conditions in two different bioreactors

The murine B-lymphocyte hybridoma cell line, CC9C10, was grown in serum-free continuous culture at steady-state dissolved oxygen (DO) concentrations of 10%, 50%, and 100% of air saturation in both LH Series 210 (LH) and New Brunswick Scientific (NBS) CelliGen bioreactors. All culture parameters were monitored and controlled and were nominally identical at steady state in the two bioreactors. The secreted monoclonal antibody (mAb), an immunoglobulin G(1), was purified and subjected to enzymatic deglycosylation using peptide N-glycosidase F (PNGase F). Asparagine-linked (N-linked) oligosaccharide pools released from mAb samples cultured in each bioreactor at each of the three DO setpoints were analyzed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The predominant N-linked structures were core-fucosylated asialo biantennary chains with varying galactosylation. There were also minor amounts of monosialyl oligosaccharides and trace amounts of afucosyl oligosaccharides. The level of DO affects the glycosylation of this mAb. A definite reduction in the level of galactosylation of N-glycan chains was observed at lower DO in both bioreactors, as evidenced by prominent increases in the relative amounts of agalactosyl chains and decreases in the relative amounts of digalactosyl chains-with the relative amounts of monogalactosyl chains being comparatively constant. However, the quantitative results are not precise matches between the two bioreactors. The effect of DO on galactosylation is less pronounced in the NBS bioreactor than in the LH bioreactor, particularly the shift between the relative amounts of agalactosyl and digalactosyl chains in 10% and 50% DO. There are also perceptibly higher levels of sialylation of the mAb glycans in the NBS bioreactor than in the LH bioreactor at all three DO setpoints. The results indicate that the DO effect is not bioreactor specific and that nominally identical steady-state conditions in different chemostat bioreactors may still lead to some incongruities in glycosylation, possibly due to the particular architectures of the bioreactors and the design of their respective monitoring and control systems. The observed differences in N-linked glycosylation of the mAb secreted by the hybridoma grown in the LH and NBS bioreactors may be explained by the differences in oxygen supply and control strategies between the two bioreactors.     

Bioreactor design for propagation of somatic embryos

Six identical bioreactors were constructed and built at the Agricultural University of Norway to provide optimal conditions for plant cell regeneration from cells into somatic embryos (clonal or somatic seeds). This was made possible through cooperation in COST87 by a European network in a working group on regeneration from plant cell cultures. The bioreactor design provides gentle stirring through a slow-speed stirrer that regularly changes direction of rotation to prevent quiet zones in the suspension in which cells can settle and grow. In addition, the oxygen is provided, bubble-free, through thin silicone tubing loops that are hanging loose, moving with the liquid to prevent cell growth on these tubes. We used off-the-shelf components whenever possible, to reduce the costs to a minimum, which was another aim of the construction. The result was a suite of relatively inexpensive computer-controlled bioreactors that could control temperature, oxygen, pH, stirrer speed and stirrer direction. In addition, we have provided different light spectral qualities by simple means of filtering the light. Using the present software, the parameters can be set up to alter every hour during the 24-h day/night cycle. All our cultures have improved growth in the bioreactors compared to identical cultures in Erlenmeyer flasks. The cultures used are: embryogenic cultures of carrot (Daucus carota), Norway spruce (Picea abies), birch (Betula pendula), cyclamen (Cyclamen persicum) and shoot cultures of Christmas begonia (Begonia x cheimantha). The paper also discusses recommendations for improvements of the current system for future revisions.     

Application of bioreactor design principles to plant micropropagation

Principles of oxygen consumption, oxygen transport, suspension, and mixing are discussed in the context of propagating aggregates of plant tissue in liquid suspension bioreactors. Although micropropagated plants have a relatively low biological oxygen demand (BOD), the relatively large tissue size and localization of BOD in meristematic regions will typically result in oxygen mass transfer limitations in liquid culture. In contrast to the typical focus of bioreactor design on gas–liquid mass transfer, it is shown that media-solid mass transfer limitations limit oxygen available for aerobic plant tissue respiration. Approaches to improve oxygen availability through gas supplementation and bioreactor pressurization are discussed. The influence of media components on oxygen availability are also quantified for plant culture media. Experimental studies of polystyrene beads in suspension in a 30-l air-lift and stirred bioreactors are used to illustrate design principles for circulation and mixing. Potential limitations to the use of liquid suspension culture due to plant physiological requirements are acknowledged.     

Computational fluid dynamics analysis of mixing and gas–liquid mass transfer in wave bag bioreactor

Single use bioreactors provide an attractive alternative to traditional deep-tank stainless steel bioreactors in process development and more recently manufacturing process. Wave bag bioreactors, in particular, have shown potential applications for cultivation of shear sensitive human and animal cells. However, the lack of knowledge about the complex fluid flow environment prevailing in wave bag bioreactors has so far hampered the development of a scientific rationale for their scale up. In this study, we use computational fluid dynamics (CFD) to investigate the details of the flow field in a 20-L wave bag bioreactor as a function of rocking angle and rocking speed. The results are presented in terms of local and mean velocities, mixing, and energy dissipation rates, which are used to create a process engineering framework for the scale-up of wave bag bioreactors. Proof-of-concept analysis of mixing and fluid flow in the 20-L wave bag bioreactor demonstrates the applicability of the CFD methodology and the temporal and spatial energy dissipation rates integrated and averaged over the liquid volume in the bag provide the means to correlate experimental volumetric oxygen transfer rates (k_La) data with power per unit volume. This correlation could be used as a rule of thumb for scaling up and down the wave bag bioreactors.