Alvin W. Nienow

Physiological responses to mixing in large scale bioreactors

Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

On impeller circulation and mixing effectiveness in the turbulent flow regime

Impeller effectiveness has often been evaluated via either mixing time, θm, or flow number, Fl; and a direct connection between the two has also been assumed in some cases. Here, these concepts are considered in the light of recent theoretical and experimental work. It is shown that an equation for mixing time recently published by BHR Group can be related to a basic turbulence model. It is also shown that this equation is superior to those based on a theory linking mixing time to the flow generated by the impeller. The new equation and theory implies that all impeller types of equal impeller-to-tank diameter ratio are equally energy efficient in achieving overall homogenisation. On the other hand, impeller efficiency based on the flow generated, as measured by laser Doppler anemometry at equal measured power, suggests a significant difference between impeller types.

The effects of agitation intensity with and without continuous sparging on the growth and antibody production of hybridoma cells

Abstract The response of 3 murine hybridomas to increasing speeds of agitation from 100 to 450 rpm with propellers and Rushton turbines in surface-aerated bioreactors of 1.4 l has been determined with pO 2 being kept above a minimum level of 20%. Cell growth and viability, antibody production, glucose consumption, lactate production and metabolic activity have been measured and found to be unaffected over this range of speeds. Scanning electron micrographs (SEM) showed no detectable cell damage even at the highest intensity of agitation. However, once the culture was continuously sparged with air the net cell growth rate and the maximum cell number fell markedly, that fall increasing with increasing agitator speed. To generalise these findings and compare them with literature reports, the implications of this speed range on the turbulence parameters have been discussed. The implications for scale-up are also considered.

Mixing in large-scale vessels stirred with multiple radial or radial and axial up-pumping impellers: modelling and measurements

Mixing phenomena are regarded as one of the major factors responsible for the failure to successfully scale up some bioprocesses. Such phenomena have been investigated within the framework of an EC project ‘Bioprocess Scale-up Strategy’. Mixing in bioreactors depends on energy input, impeller type, reactor configuration and impeller geometry. Here, two different reactors of volumes 12 and 30m3 were used, and they were equipped with either multiple Rushton turbines or with a combination of a Scaba 6SRGT radial impeller with multiple 3SHP axial up-pumping hydrofoils above it. Mixing time, power consumption, gas hold-up and liquid velocities were measured at different stirrer speeds and aeration rates in water. At the same total specific power input, aeration did not influence the mixing time much unless it changed the bulk flow pattern. A considerable reduction of mixing time was achieved if the upper impellers were axial instead of radial Rushtons at the same power consumption. The improvement with the axial impellers could be related to the reduction of axial flow barriers due to different circulation flow patterns. The Compartment Model Approach (CMA) was used to develop a flow model based on the general knowledge of the hydrodynamics of both unaerated and aerated stirred vessels. The model was successfully verified for different impeller and reactor configurations and different scales with measured pulse response curves, using either a fluorescent or a hot water tracer. The model can be used for process design purposes.

Dependence of mycelial morphology on impeller type and agitation intensity.

The influence of the agitation conditions on the morphology of Penicillium chrysogenum (freely dispersed and aggregated forms) was examined using radial (Rushton turbines and paddles), axial (pitched blades, propeller, and Prochem Maxflow T), and counterflow impellers (Intermig). Culture broth was taken from a continuous fermentation at steady state and was agitated for 30 min in an ungassed vessel of 1.4‐L working volume. The power inputs per unit volume of liquid in the tank, P/VL, ranged from 0.6 to 6 kW/m3. Image analysis was used to measure mycelial morphology. To characterize the intensity of the damage caused by different impellers, the mean total hyphal length (freely dispersed form) and the mean projected area (all dispersed types, i.e., also including aggregates) were used. [In this study, breakage of aggregates was taken into account quantitatively for the first time.]

On the Sauter mean diameter and size distributions in turbulent liquid/liquid dispersions in a stirred vessel

Abstract It has been customary to relate the Sauter mean drop size, d 32 , to Weber number, We , by the relationship d 32 D∝We -0.6 . This relationship comes from the assumptions that d 32 is a constant fraction of d max , where d max is the maximum stable drop size and that d max can be predicted from theoretical considerations if Kolmogoroffs theory of isotropic turbulence is used for estimating the disruptive forces. Here it is shown, firstly on theoretical grounds, that the assumption d 32 ∝ d max is not justified; and secondly that experimental results from 18 different runs neither support d 32 = Ad max where A is system and agitation conditions independent nor that the exponent on We is −0.6. Further, though cumulative volume size distributions indicate self-similarity, as suggested previously, the number probability density distributions show strong bi-modality at low speed and low dispersed-phase concentration which lessens with increasing concentration and becomes uni-modal at high speeds. This study suggests that in spite of the great deal of work which has already been done, more is required in which the relationship between mean drop size and drop size distributions and agitation conditions over a wider range of concentrations are further investigated.

The dependency on scale of power numbers of Rushton disc turbines

Extensive and very accurate power numbers have been obtained for a wide range of Rushton disc turbines using water as the working fluid in six fully baffled vessels from 0.22 to 1.83 m diameter. The results show that for Reynolds numbers ⩾ 2 × 104, the average peak power number Po is dependent on the disc thickness (χ1) to impeller diameter (D) ratio and to the vessel diameter (T). Provided χ1/D is constant, Po is independent of impeller to vessel ratio in the range 0.25 ⩾ D/T ⩽ 0.70. For 0.01 ⩽ χ1/D ⩽ 0.05 and for the range of vessels studied, the equation fitted the data to within ± 3% where To is a 1 m diameter vessel. An F-test shows that the inclusion of the small effect of scale, (T/To), is statistically significant at the 99% level. The implications of these scale and geometrical effects for mixing research, process design and scale-up are discussed.

The scale-up of microbial batch and fed-batch fermentation processes

Publisher Summary The scale-up of single-celled aerobic microbial fermentation processes is complicated that can lead to unpredictable process performance. However, this is not due to the introduction of fluid dynamic generated stresses (or so-called “shear damage”), whether arising from agitator generated turbulence or bursting bubbles, rather it is because the large-scale fed-batch bioreactor provides a very dynamic environment with large spatial and temporal heterogeneities. Such environmental heterogeneities can induce multiple physiological responses in cells. These responses consume energy and resources such that biomass concentration and product yields can be reduced. These phenomena are not observed in well-mixed homogeneous laboratory-scale reactors where much process development is done and their effects are difficult to model mathematically. Therefore, the ability to obtain data on how a recombinant laboratory process may perform at the large scale, dependent on feeding regime employed or controlling action taken is invaluable for any detailed and informed development program.

The translation of cell-based therapies: clinical landscape and manufacturing challenges

Cell-based therapies have the potential to make a large contribution toward currently unmet patient need and thus effective manufacture of these products is essential. Many challenges must be overcome before this can become a reality and a better definition of the manufacturing requirements for cell-based products must be obtained. The aim of this study is to inform industry and academia of current cell-based therapy clinical development and to identify gaps in their manufacturing requirements. A total of 1342 active cell-based therapy clinical trials have been identified and characterized based on cell type, target indication and trial phase. Multiple technologies have been assessed for the manufacture of these cell types in order to facilitate product translation and future process development.

Bubble sizes in electrolyte and alcohol solutions in a turbulent stirred vessel

Bubble size distributions have been measured by a new video technique at 3 points near the wall in a vessel of 150 mm diameter air-sparged at ∼1 vvm agitated by a Rushton turbine at an energy dissipation rate of ∼1Wkg-1. Water and solutions of electrolytes and alcohols were used. These solutes give surface tensions less than water (alcohols) and greater than water (electrolytes) and concentrations were chosen to produce solutions which, based on work in bubble columns and coalescence cells, can be considered partially-coalescing and non-coalescing. Regardless of surface tension, the bubble sizes in the non-coalescing solutions were approximately the same and much less than water, whilst those in the partiallycoalescing case where the surface tension was approximately equal to that of water, gave intermediate sizes. Thus, the Weber number cannot correlate such results. On the other hand, the concept of bubble sizes being controlled by coalescence-inhibitionafter initial break-up works well. In all cases, bubbles as small as 40 μm were found and even in water some 40% were below 300 μm, the smallest size practicably measurable by a capillary technique. Surprisingly, the bubble size decreased with vessel height and possible reasons for this are discussed.