Integrating a 250 mL-spinner flask with other stirred bench-scale cell culture devices: a mass transfer perspective

The bioprocess development cycle is a complex task that requires a complete understanding of the engineering of the process (e.g., mass transfer, mixing, CO(2) removal, process monitoring, and control) and its affect on cell biology and product quality. Despite their widespread use in bioprocess development, spinner flasks generally lack engineering characterization of critical physical parameters such as k(L)a, P/V, or mixing time. In this study, mass transfer characterization of a 250-mL spinner flask using optical patch-based sensors is presented. The results quantitatively show the effect of the impeller type, liquid filling volume, and agitation speed on the volumetric mass transfer coefficient (k(L)a) in a 250-mL spinner flask, and how they can be manipulated to match mass transfer capability at large culture devices. Thus, process understanding in spinner flasks can be improved, and these devices can be seamlessly integrated in a rational scale-up strategy from cell thawing to bench-scale bioreactors (and beyond) in biomanufacturing.     

Transport phenomena, reactor design and scale-up

This review will cover the area of impeller-mixed stirred-tank reactors. In addition, it will consider bubble columns, in which air or gas is passed up a liquid filled column through distribution plates covering the full area of the column, and also airlift reactors, in which the air is confined in a channel by means of a loop or draft tube designed to impart a certain type of overall circulatory pattern to the entire tank.There is considerable interest in the kinetics inside the solid part of various kinds of immobilized solid pellet type of enzymes and catalysts. The use of these particles in fixed bed reactors is also covered.     

Verfahrenstechnische Grundlagen der Reaktorgestaltung

Starting with the definition of the process term k_La, steady state and nonsteady state measuring methods are described for its determination. Then the sorption characteristics for mixing vessels and for bubble columns are presented with respect to the coalescence behaviour of the system treated. They permit the scale-up of these devices and the optimization of their process parameters for a required oxygen uptake. In addition to the sorption characteristics for the given system the knowledge of the flooding point and the power characteristics is necessary for the lay-out of mixing vessels, whereas in the case of bubble columns the gas hold-up characteristic needs to be known.     

Survival of,and plasmid transfer between modified Enterobacter cloacae strains under long-term starvation conditions in a continuous-flow system

Continuous-flow, packed-bed column reactors, which provide an experimental model of a soil profile, were used to investigate survival of, and plasmid transfer between, strains of Enterobacter cloacae. When columns, inoculated with nutrient-sufficient donor and recipient strains, were provided with a minimal salts medium with no added carbon source, transconjugant cells appeared in their effluents. During the first few days of such experiments, the concentration of cells in the effluent declined but then the donor population stabilized, while the recipient and transconjugant populations continued to decrease. The results indicate that the amount of nutrient required to maintain and transfer plasmids is very low. No transconjugants were observed in the effluent from columns inoculated with pre-starved donor and recipient strains.     

Studies on neomycin production using immobilized cells of<Emphasis Type="Italic">S marinensis</Emphasis> NUV-5 in various reactor configurations: A technical note

Conclusion  Stirred tank, fluidized bed, and airlift reactors produced similar neomycin activity with immobilized cells. Packed bed reactor clearly under performed, probably because of insufficient aeration or mixing. Neomycin production using immobilized cells in fermentors requires good mixing and aeration.     

Acceleration of mass transfer in methane-producing loop reactors

Gas bubbles entrapped in methanogenic granules subjected to hydrostatic pressure oscillations during recirculation in loop reactors will induce intraparticle liquid flows, and thereby enhance mass transfer in excess of diffusion. This breathing particle concept was clearly demonstrated in a well defined inorganic model system. The experimental results could be described satisfactory with a structured mathematical model, while a 30% improvement is predicted for methanogenic loop reactors as compared to constant pressure systems. It is concluded that acceleration of mass transfer in gas-producing systems offers challenging perspectives for both heterogeneous catalysis and biological fermentations.     

Beyond heuristics: CFD-based novel multiparameter scale-up for geometrically disparate bioreactors demonstrated at industrial 2kL–10kL scales

The timely delivery of the most up-to-date medicines and drug products is essential for patients throughout the world. Successful scaling of the bioreactors used within the biopharmaceutical industry plays a large part in the quality and time to market of these products. Scale and topology differences between vessels add a large degree of complication and uncertainty within the scaling process. Currently, this approach is primarily achieved through extensive experimentation and facile empirical correlations, which can be costly and time consuming while providing limited information. The work undertaken in the current study demonstrates a more robust and complete approach using computational fluid dynamics (CFD) to provide potent multiparameter scalability, which only requires geometric and material properties before a comprehensive and detailed solution can be generated. The CFD model output parameters that can be applied in the scale-up include mass transfer rates, mixing times, shear rates, gas hold-up values, and bubble residence times. The authors examined three bioreactors with variable geometries and were able to validate them based on single-phase and multiphase experiments. Furthermore, leveraging the resulting CFD output information enabled the authors to successfully scale-up from a known 2kL to a novel and disparate 5kL single-use bioreactor in the first attempted cell culture. This multiparameter scaling approach promises to ultimately lead to a reduction in the time to market providing patients with earlier access to the most groundbreaking medicines.     

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.     

Monitoring gradient formation in a jet aerated bioreactor

Jet aerated loop reactors (JLRs) provide high mass transfer coefficients (k_La) and can be used for the intensification of mass transfer limited reactions. The jet loop reactor achieves higher k_La values than a stirred tank reactor (STR). The improvement relies on significantly higher local power inputs (～10⁴) than those obtainable with the STR. Operation at high local turnover rates requires efficient macromixing, otherwise reactor inhomogeneities might occur. If sufficient homogenization is not achieved, the selectivity of the reaction and the respective yields are decreased. Therefore, the balance between mixing and mass transfer in jet loop reactors is a critical design aspect. Monitoring the dissolved oxygen levels during the turnover of a steady sodium sulfite feed implied the abundance of gradients in the JLR. Prolonged mixing times at identical power input and aeration rates (～100%) were identified for the JLR in comparison to the STR. The insertion of a draft tube to the JLR led to a more homogenous dissolved oxygen distribution, but unfortunately a reduction of mixing time was not achieved. In case of increased medium viscosities as they may arise in high cell density cultivations, no gradient formation was detected. However, differences in medium viscosity significantly altered the mass transfer and mixing performance of the JLR.     

Predispersed solvent extraction of succinic acid aqueous solution by colloidal liquid aphrons in column

A study of the PDSE (predispersed solvent extraction) for succinic acid by colloidal liquid aphrons was conducted. The organic phase contaning TOA (tri-n-octylamine) and 1-octanol permits a selective extraction of succinic acid from its aqueous solution. There was no difference of the extractability of PDSE and that of conventional mixer-settler type extraction. Taking into account the no mechanical mixing in PDSE, it was concluded that the PDSE process is more adaptive than the conventional mixer-settler type extraction process. From mass transfer analysis at the various concentration of TOA in counter-current continuous operation, the concentration of TOA had no influence on the mass transfer coefficient. The loading values in continuous PDSE were almost same as those in batch operation.     

A simple and inexpensive on-column frit fabrication method for fused-silica capillaries for increased capacity and versatility in LC-MS/MS applications

A modified sol-gel method for a one-step on-column frit preparation for fused-silica capillaries and its utility for peptide separation in LC-MS/MS is described. This method is inexpensive, reproducible, and does not require specialized equipments. Because the frit fabrication process does not damage polyimide coating, the frit-fabricated column can be tightly connected on-line for high pressure LC. These columns can replace any capillary liquid transfer tubing without any specialized connections up-stream of a spray tip column. Therefore multiple columns with different phases can be connected in series for one- or multiple-dimensional chromatography.     

Mathematical Modeling of Size Exclusion Chromatography

A mathematical model of the size exclusion chromatography (SEC) process in chromatographic columns has been developed. It considers the following three mass transfer processes in the SEC column: axial dispersion in the bulk‐fluid phase, interfacial film mass‐transfer between the stationary and mobile phases, and diffusion of solutes within the macro pores of the packing particles. Differential equations of the process model were solved by the finite difference method. Characteristics of the column and the packing particles (bed void volume fraction, particle porosity, accessible particle porosity) were obtained experimentally, as well as retention times of different molecules with known molecular weights. Experiments were performed with two different columns containing two different packing materials, Superdex 75 HR 10/30 and BioSep SEC S2000, respectively. The model has been validated by comparing theoretical and experimental retention times for the different columns.     

Mass transfer limitation of microbial growth and pollutant degradation

Organic pollutants in soil can be removed by biotechnological treatment. A limitation of this technology is the efficiency of biodegradation. In many cases, the bulk of the pollution can be removed but residual pollutants remain and biodegradation rates are slower than expected from laboratory trials. Low biodegradation rates are often a result of limited accessibility of the pollutants. Major reasons for the reduced bioavailability are the unequal spatial distribution of microorganisms and pollutants and the retardation of substrate diffusion by the soil matrix. Mechanical mixing and the addition of surfactants are possible approaches to improve the bioavailability of pollutants during bioremediation. The application of flow-stop-flow techniques may be of help to overcome the limitations resulting from advective-diffusive transport mechanisms during pump-and-treat remediation of contaminant plumes. Received 31 October 1995/ Accepted in revised form 31 March 1996     

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.     

Enhancing the functionality of a microscale bioreactor system as an industrial process development tool for mammalian perfusion culture

Without a scale-down model for perfusion, high resource demand makes cell line screening or process development challenging, therefore, potentially successful cell lines or perfusion processes are unrealized and their ability untapped. We present here the refunctioning of a high-capacity microscale system that is typically used in fed-batch process development to allow perfusion operation utilizing in situ gravity settling and automated sampling. In this low resource setting, which involved routine perturbations in mixing, pH and dissolved oxygen concentrations, the specific productivity and the maximum cell concentration were higher than 3.0 × 10⁶ mg/cell/day and 7 × 10 ⁷ cells/ml, respectively, across replicate microscale perfusion runs conducted at one vessel volume exchange per day. A comparative analysis was conducted at bench scale with vessels operated in perfusion mode utilizing a cell retention device. Neither specific productivity nor product quality indicated by product aggregation (6%) was significantly different across scales 19 days after inoculation, thus demonstrating this setup to be a suitable and reliable platform for evaluating the performance of cell lines and the effect of process parameters, relevant to perfusion mode of culturing.     

动物细胞培养用生物反应器设计原理

动物细胞培养用生物反应器设计和放大的关键问题是细胞破损与供氧和混合的矛盾,在分析细胞破损机理基础上,提出了动物细胞培养生物反应器的设计原理——设计模型和有关设计条件,从而清楚地确立了细胞死亡速度与培养基组成、反应器设计和操作参数间的定量关系,以及反应器设计应遵循的保证细胞生长和满足传质要求的条件。还对强化传质和抑制细胞破损这一矛盾作了简要分析和讨论。     

Liquid circulation and mixing in tower fermenters with recirculation loop

Liquid circulation and mixing time studies were carried out in a laboratory bubble column and modified bubble columns. Liquid circulation and mixing time are both strongly dependent on the geometrical configuration of the reactor. Internal recycle loop and external recycle loop shorten the mixing time compared to the bubble column.     

Outgassing Losses of Toluene and m-Xylene Evaluated by 14C-Based Mass Balances for Laboratory Bioventing Simulations

Mixtures of toluene, ethylbenzene, and the xylenes spiked with ¹⁴C-labeled toluene or m-xylene were added to bench-scale bioventing simulation columns filled with hydrocarbon-contaminated subsurface soils. After 2 to 4 weeks of incubation during which air was pumped through the column at rates of at least 2?ml·min^?1·kg^?1 between 54 and 84% of the radiolabel was recovered in traps as outgassed parent compound from four columns sterilized with gamma-irradiation. In contrast, seven nonsterilized but otherwise identically treated (except for inorganic nitrogen addition) columns lost less than 0.4% (and one column lost 0.7%) of the radiolabel through outgassing of the parent compound. Nonsterilized columns lost 40 to 61% of the radiolabel as ¹⁴CO₂, whereas gamma-irradiated columns usually lost only trace amounts of ¹⁴C in this form. Biologically active columns also retained much larger fractions than sterilized columns of the radiolabel in the subsoil in forms, possibly microbial biomass, from which it could be recovered by wet oxidation. Addition of 10 or 40?mg/kg of mineral nitrogen had no consistent effect on bioventing performance.     

Hydrodynamics, mass transfer, and yeast culture performance of a column bioreactor with ejector

A bubble column fitted with an ejector has been tested for its physical and biological performance. The axial diffusion coefficient of the liquid phase in the presence of electrolytes and ethanol was measured by a stimulus-response technique with subsequent evaluation by means of a diffusion model. In contrast to ordinary bubble columns, the coefficient of axial mixing is inversely dependent on the superficial air velocity. The liquid velocity acts in an opposite direction to the backmixing flow in the column. The measurement of volumetric oxygen transfer coefficient in the presence of electrolytes and ethanol was performed using a dynamic gassing-in method adapted for a column. The data were correlated with the superficial air and liquid velocities, total power input, and power for aeration and mixing; the economy coefficient of oxygen transfer was used for finding an optimum ratio of power for aeration and pumping. Growth experiments with Candida utilis on ethanol confirmed some of the above results. Biomass productivity of 2.5 g L(-1) h(-1) testifies about a good transfer capability of the column. Columns fitted with pneumatic and/or hydraulic energy input may be promising for aerobic fermentations considering their mass transfer and mixing characteristics.     

Microbiological aspects of surfactant use for biological soil remediation

Biodegradation of hydrophobic organic compounds in polluted soil is a process involving interactions among soil particles, pollutants, water, and micro-organisms. Surface-active agents or surfactants are compounds that may affect these interactions, and the use of these compounds may be a means of overcoming the problem of limited bioavailability of hydrophobic organic pollutants in biological soil remediation. The effects of surfactants on the physiology of micro-organisms range from inhibition of growth due to surfactant toxicity to stimulation of growth caused by the use of surfactants as a co-substrate. The most important effect of surfactants on the interactions among soil and pollutant is stimulation of mass transport of the pollutant from the soil to the aqueous phase. This can be caused by three different mechanisms: emulsification of liquid pollutant, micellar solubilisation, and facilitated transport. The importance of these mechanisms with respect to the effect of surfactants on bioavailability is reviewed for hydrophobic organic pollutants present in different physical states. The complexity of the effect of surfactants on pollutant bioavailability is reflected by the results in the literature, which range from stimulation to inhibition of desorption and biodegradation of polluting compounds. No general trends can be found in these results. Therefore, more research is necessary to make the application of surfactants a standard tool in biological soil remediation. This revised version was published online in August 2006 with corrections to the Cover Date.