Studies supporting the use of mechanical mixing in large scale beer fermentations

Brewing fermentations have traditionally been undertaken without the use of mechanical agitation, with mixing being provided only by the fluid motion induced by the CO₂ evolved during the batch process. This approach has largely been maintained because of the belief in industry that rotating agitators would damage the yeast. Recent studies have questioned this view. At the bench scale, brewer’s yeast is very robust and withstands intense mechanical agitation under aerobic conditions without observable damage as measured by flow cytometry and other parameters. Much less intense mechanical agitation also decreases batch fermentation time for anaerobic beer production by about 25% compared to mixing by CO₂ evolution alone with a small change in the concentration of the different flavour compounds. These changes probably arise for two reasons. Firstly, the agitation increases the relative velocity and the area of contact between the cells and the wort, thereby enhancing the rate of mass transfer to and from the cells. Secondly, the agitation eliminates spatial variations in both yeast concentration and temperature, thus ensuring that the cells are maintained close to the optimum temperature profile during the whole of the fermentation time. These bench scale studies have recently been supported by results at the commercial scale from mixing by an impeller or by a rotary jet head, giving more consistent production without changes in final flavour. It is suggested that this reluctance of the brewing industry to use (adequate) mechanical agitation is another example where the myth of shear damage has had a detrimental effect on the optimal operation of commercial bioprocessing.     

Temperature control in a continuously mixed bioreactor for solid-state fermentation

A continuously mixed, aseptic paddle mixer was used successfully for solid-state fermentation (SSF) with Aspergillus oryzae on whole wheat kernels. Continuous mixing improved temperature control and prevented inhomogeneities in the bed. Respiration rates found in this system were comparable to those in small, isothermal, unmixed beds, which showed that continuous mixing did not cause serious damage to the fungus or the wheat kernels. Continuous mixing improves heat transport to the bioreactor wall, which reduces the need for evaporative cooling and thus may help to prevent the desiccation problems that hamper large-scale SSF. However, scale-up calculations for the paddle mixer indicated that wall cooling becomes insufficient at the 2-m(3) scale for a rapidly growing fungus like Aspergillus oryzae. Consequently, evaporative cooling will remain important in large-scale mixed systems. Experiments showed that water addition will be necessary when evaporative cooling is applied in order to maintain a sufficiently high water activity of the solid substrate. Mixing is necessary to ensure homogeneous water addition in SSF. Automated process control might be achieved using the enthalpy balance. The enthalpy balance for the case of evaporative cooling in the paddle mixer was validated. This work shows that continuous mixing provides promising possibilities for simultaneous control of temperature and moisture content in solid-state fermentation on a large scale.     

Influence of bioreactor hydraulic characteristics on a Saccharomyces cerevisiae fed-batch culture: hydrodynamic modelling and scale-down investigations

Yeast is a widely used microorganism at the industrial level because of its biomass and metabolite production capabilities. However, due to its sensitivity to the glucose effect, problems occur during scale-up to the industrial scale. Hydrodynamic conditions are not ideal in large-scale bioreactors, and glucose concentration gradients can arise when these bioreactors are operating in fed-batch mode. We have studied the effects of such gradients in a scale-down reactor, which consists of a mixed part linked to a non-mixed part by a recirculation pump, in order to mimic the hydrodynamic conditions encountered at the large scale. During the fermentation tests in the scale-down reactor, there was a drop in both biomass yield (ratio between the biomass produced and the glucose added) and trehalose production and an increase in both fermentation time (time between inoculation and beginning of stationary phase) and ethanol production. We have developed a stochastic model which explains these effects as the result of an induction process determined mainly by the hydrodynamic conditions. The concentration profiles experienced by the microorganisms during the scale-down tests were expressed and linked to the biomass yields of the scale-down tests.     

Some problems of industrial scale-up

There are problems associated with all stages of the scaling-up of biological processes developed on a laboratory scale. The large size of industrial fermenters required for commercial viability (up to 100 m³) gives rise to problems of mixing, heat transfer, and control as well as problems in physically handling large volumes of liquids and gases. The concentration of products obtained from biological processes is low (typically 10 per cent), so recovery processes require a large energy input. The optimization and integration of all parts of the process, from the initial fermentation to marketable product, is essential.     

Prelude to rational scale-up of penicillin production: a scale-down study

Penicillin is one of the best known pharmaceuticals and is also an important member of the β-lactam antibiotics. Over the years, ambitious yields, titers, productivities, and low costs in the production of the β-lactam antibiotics have been stepwise realized through successive rounds of strain improvement and process optimization. Penicillium chrysogenum was proven to be an ideal cell factory for the production of penicillin, and successful approaches were exploited to elevate the production titer. However, the industrial production of penicillin faces the serious challenge that environmental gradients, which are caused by insufficient mixing and mass transfer limitations, exert a considerably negative impact on the ultimate productivity and yield. Scale-down studies regarding diverse environmental gradients have been carried out on bacteria, yeasts, and filamentous fungi as well as animal cells. In accordance, a variety of scale-down devices combined with fast sampling and quenching protocols have been established to acquire the true snapshots of the perturbed cellular conditions. The perturbed metabolome information stemming from scale-down studies contributed to the comprehension of the production process and the identification of improvement approaches. However, little is known about the influence of the flow field and the mechanisms of intracellular metabolism. Consequently, it is still rather difficult to realize a fully rational scale-up. In the future, developing a computer framework to simulate the flow field of the large-scale fermenters is highly recommended. Furthermore, a metabolically structured kinetic model directly related to the production of penicillin will be further coupled to the fluid flow dynamics. A mathematical model including the information from both computational fluid dynamics and chemical reaction dynamics will then be established for the prediction of detailed information over the entire period of the fermentation process and thereby for the optimization of penicillin production, and subsequently also benefiting other fermentation products.     

The use of yeast inoculation in fermentation for port production; effect on total potential ethyl carbamate

Summary A commercial wine yeast Saccharomyces cerevisiae UCD 522 (pre-cultured in the presence of certain mass-labelled amino acids) was inoculated into a port must which was then allowed to ferment under controlled conditions of temperature and agitation. The influence of potential ethyl carbamate (EC) precursor formed due to yeast pre-culture, upon total potential EC levels was studied at various stages of fermentation. Pre-culture accumulation did not give rise to detectable levels of EC precursor during port fermentation.     

Characterization and feasibility of a miniaturized stirred tank bioreactor to perform E. coli high cell density fed-batch fermentations

The use of small scale bioreactors that are mechanically and functionally similar to large scale reactors is highly desirable to accelerate bioprocess development because they enable well-defined scale translations. In this study, a 25-mL miniaturized stirred tank bioreactor (MSBR) has been characterized in terms of its power input, hydrodynamics, and volumetric oxygen transfer coefficient (k(L)a) to assess its potential to grow high cell density (HCD) cultures using adequate scale-down criteria. Engineering characterization results show scale down, based on matched specific power input (P(G)/V), is feasible from a 20-L pilot scale stirred tank bioreactor. Results from fed-batch fermentations performed using Fab' producing E. coli W3110 at matched (P(G)/V) in the MSBR and 20-L STR demonstrated that the MSBR can accurately scale down the 20-L fermentation performance in terms of growth and Fab' production. Successful implementation of a fed-batch strategy in the MSBR resulted in maximum optical density of ca. 114 and total Fab' concentration of 940 μg/mL compared with ca. 118 and 990 μg/mL in 20-L STR. Furthermore, the use of the MSBR in conjunction with primary recovery scale-down tools to assess the harvest material of both reactors showed comparable shear sensitivity and centrifugation performance. The conjoint use of the MSBR with ultra scale-down (USD) centrifugation mimics can provide a cost-efficient manner in which to design and develop bioprocesses that account for good upstream performance as well as their manufacturability downstream.     

Low-pressure airlift fermenter for single cell protein production: II. Continuous culture of pichia yeast

Experiments using Pichia yeast grown on n-paraffins have been conducted in laboratory 10-L airlift fermenters and in a 640-L module of commercial scale. Results confirmed the design concept of combining oxygen transfer and fermenter cooling with low-pressure air. However, in the absence of mass transport constraints, the build up of toxic factors in the fermenter appeared to be a major variable limiting cell productivity. Foaming in the large fermenter also presented a serious problem, which must be solved before low-pressure airlift fermenters become practical.     

The use of fluorometry for on-line measurement of mixing time and hold-up in fermentations

Summary Mixing times and gas hold-ups in a 250L bioreactor containing a phosphate buffer or an active fermentation were determined on-line using fluorometric (MEFS and NADH) probes as functions of agitation and aeration rates. Both mixing time and hold-up of a fermentaion can be determined using a MEFS probe. Hold-up may also be measured with a presently available commercial NADH probe.     

Scale up of a viscous fungal fermentation: application of scale-up criteria with regime analysis and operating boundary conditions

The scale up of the novel, pharmaceutically important pneumocandin (B(0)), from the filamentous fungus Glarea lozoyensis was successfully completed from pilot scale (0.07, 0.8, and 19 m(3)) to production scale (57 m(3)). This was accomplished, despite dissimilar reactor geometry, employing a combination of scale-up criteria, process sensitivity studies, and regime analysis using characteristic time constants for both oxygen mass transfer and bulk mixing. Dissolved oxygen tension, separated from the influence of agitation by gas blending at the 0.07 m(3)-scale, had a marked influence on the concentrations of pneumocandin analogs with different levels of hydroxylation, and these concentrations were used as an indicator of bulk mixing upon scale up. The profound impact of dissolved oxygen tension (DOT) (low and high levels) on analog formation dictated the use of constant DOT, at 80% air saturation, as a scale-up criterion. As a result k(L)a, Oxygen uptake rate (OUR) and hence the OTR were held constant, which were effectively conserved across the scales, while the use of other criterion such as P(g)/V(L), or mixing time were less effective. Production scale (57 m(3)) mixing times were found to be faster than those at 19 m(3) due to a difference in liquid height/tank diameter ratio (H(L)/D(T)). Regime analysis at 19 and 57 m(3) for bulk mixing (t(c)) and oxygen transfer (1/k(L)a) showed that oxygen transfer was the rate-limiting step for this highly shear thinning fermentation, providing additional support for the choice of scale-up criterion.     

Solid substrate fermentation of wheat straw to fungal protein

Steam-treated wheat straw at a 70% (w/w) moisture level was subjected to solid substrate fermentation (SSF) with Trichoderma reesei (Riga, USSR) or a mixed culture of T. reesei and Endomycopsis fibuliger (R-574) in fermentation equipment of various design: some with mixing, some with stationary layers, including a mixedlayer 1.5-m(3) pilot plant scale fermenter. The best protein productivity was obtained in stationary layer fermenters with a product containing 13% protein. The main limitations of lignocellulose SSF, such as hindrance of fungal growth, limiting accessibility and availability of substrate, and difficulty in moisture and heat control, were analyzed. The technological parameters of SSF, submerged fermentation, and alternate lignocellulose conversion processes were compared. The SSF had lower overall efficiency but higher product concentration per reaction volume than other conversion schemes.     

Optimized fermentation of grape juice by laboratory strains of Saccharomyces cerevisiae

Laboratory strains of yeast ( Saccharomyces cerevisiae ) based on S288C ferment grape juice relatively poorly. We show that slow fermentation appears to be inherent to this strain, because the original S288C isolate shows fermentation similar to current laboratory isolates. We demonstrate further that some auxotrophic mutations in the laboratory strain show reduced rates of fermentation in grape juice, with lysine auxotrophs particularly impaired compared with isogenic Lys⁺ strains. Supplementing lysine at a 10-fold higher concentration than recommended allowed yeast cultures to reach higher final cell densities and restored the fermentation rate of auxotrophic strains to those of the corresponding wild-type strains. However, even with the additional supplementation, the fermentation rates of S288C strains were still slower than those of a commercial wine yeast strain. Conditions were developed that enable auxotrophic laboratory strains derived from S288C to ferment grape juice to completion with high efficiency on a laboratory scale. Fermentation in media based on grape juice will allow the suite of molecular genetic tools developed for these laboratory strains to be used in investigations of complex ferment characteristics and products.     

A comparison of laboratory and pilot-scale fermentations in winemaking conditions

We investigated the influence of the fermenter size on alcoholic fermentation. Experiments were carried out at pilot scale, in 100-L fermenters, and at laboratory scale, in stirred and static 1-L fermenters. Two musts, Grenache blanc and Sauvignon, were fermented with and without the addition of solid particles from grape musts. Highly clarified must fermentation kinetics was strongly affected by the scale of the experiment, with slower fermentation occurring in the 100-L fermenter. Alcohol, ester, and thiol synthesis in clarified sauvignon must fermentation was also strongly correlated with the fermentation scale. Addition of solid particles from grape tended to reduce the effects on kinetics associated with increasing the scale of the fermentation, by increasing the maximum rate of CO₂ production, and by shortening the duration of fermentation. The addition of such particles also decreased the effects of scaling up the fermentation on the concentration of some volatile compounds, i.e., isoamyl acetate, ethyl octanoate, but did not decrease this effect for other compounds, such as isobutyl acetate, isobutanol, and 3-mercaptohexanol.     

Laminar-flow fluid mixer for fast fluorescence kinetics studies

The ability to mix aqueous liquids on microsecond time scales, while consuming minimal amounts of sample and maintaining UV-visible optical access to the mixing region, is highly desirable for a range of biophysical studies of fast protein and nucleic acid interactions and folding. We have constructed a laminar coaxial jet mixer that allows the measurement of UV-excited fluorescence from nanoliter and microliter quantities of material, mixed at microsecond rates. The mixer injects a narrow cylindrical stream (radius a < 1 microm) of fluorescent sample into a larger flow of diluting buffer that moves through a capillary (100 microm i.d.) at a speed approximately 20 cm/s, under laminar flow conditions (Re approximately equal to 14). Construction from a fused silica capillary allows the laser excitation (at 266 nm) and detection (at 350 nm) of tryptophan fluorescence at reasonably low working concentrations, without interference from background fluorescence. Using this mixer we have measured sub-millisecond fluorescence quenching kinetics while consuming fluorescent sample at rates no greater than 6 nl/s. Consumption of the diluting buffer is also very modest (approximately 1-3 microl/s) in comparison with other rapid mixer designs.     

Scale-up of Escherichia coli growth and recombinant protein expression conditions from microwell to laboratory and pilot scale based on matched k(L)a

Fermentation optimization experiments are ideally performed at small scale to reduce time, cost and resource requirements. Currently microwell plates (MWPs) are under investigation for this purpose as the format is ideally suited to automated high-throughput experimentation. In order to translate an optimized small-scale fermentation process to laboratory and pilot scale stirred-tank reactors (STRs) it is necessary to characterize key engineering parameters at both scales given the differences in geometry and the mechanisms of aeration and agitation. In this study oxygen mass transfer coefficients are determined in three MWP formats and in 7.5 L and 75 L STRs. k(L)a values were determined in cell-free media using the dynamic gassing-out technique over a range of agitation conditions. Previously optimized culture conditions at the MWP scale were then scaled up to the larger STR scales on the basis of matched k(L)a values. The accurate reproduction of MWP (3 mL) E. coli BL21 (DE3) culture kinetics at the two larger scales was shown in terms of cell growth, protein expression, and substrate utilization for k(L)a values that provided effective mixing and gas-liquid distribution at each scale. This work suggests that k(L)a provides a useful initial scale-up criterion for MWP culture conditions which enabled a 15,000-fold scale translation in this particular case. This work complements our earlier studies on the application of DoE techniques to MWP fermentation optimization and in so doing provides a generic framework for the generation of large quantities of soluble protein in a rapid and cost-effective manner.     

Enhancement of ribitol production during fermentation of <Emphasis Type="Italic">Trichosporonoides oedocephalis</Emphasis> ATCC 16958 by optimizing the medium and altering agitation strategies

To maximize the productivity of ribitol, which is an important starting material for the production of one expensive rare sugar, L-ribose, the effects of culture medium and agitation speed on cell growth as well as on the productivity of ribitol were thoroughly investigated in a 7 L fermentor. The maximum volumetric productivity, 0.322 g/L/h of ribitol, were obtained at an initial glucose concentration of 200 g/L in a batch culture. Based on the optimum glucose concentration, the ribitol yield conversed from glucose was up to 0.193 g/g when 1% yeast extract was used as a nitrogen source. When the agitation speed was maintained at 200 rpm, the ribitol concentration of 38.60 g/L was collected after 120 h of cultivation time. Additionally, the scheme of two-phase agitation and glucose infusion was employed. To begin, in the first 24 h of fermentation, a high agitation rate at 350 rpm and the initial glucose concentration of 50 g/L were applied, and the biomass concentration of 25.50 g/L was achieved at 36 h of incubation; whereas this value was observed until 60 h in the former batch fermentation methods. Then, in the second phase, with the agitation speed reduced to 150 rpm and the infusion amount of glucose controlled at 150 g/L, the yield of ribitol reached to 65.00 g/L in two-phase agitation fermentation and was 1.68 fold of that obtained in one-stage batch fermentation. To our knowledge, this study first demonstrates its significant effectiveness in improving ribitol production with the application of Trichosporonoides oedocephalis ATCC 16958.     

Prediction of the pilot-scale recovery of a recombinant yeast enzyme using integrated models

This article describes the rapid prediction of recovery process performance for a new recombinant enzyme product on the basis of a broad portfolio of computer models and highly targeted experimentation. A process model for the recombinant system was generated by linking unit operation models in an integrated fashion, with required parameter estimation and physical property determination accomplished using data from scale-down studies. This enabled the generic modeling framework established for processing of a natural enzyme from bakers' yeast to be applied. An experimental study of the same operations at the pilot scale showed that the process model gave a conservative prediction of recombinant enzyme recovery. The model successfully captured interactions leading to a low overall product yield and indicated the need for further study of precipitate breakage in the feed zone of a disc stack centrifuge in order to improve performance. The utility of scale-down units as an aid to fast model generation and the advantage of integrating computer modeling and scale-down studies to accelerate bioprocess development are highlighted.     

Computational fluid dynamics simulation improves the design and characterization of a plug-flow-type scale-down reactor for microbial cultivation processes

The scale-up of bioprocesses remains one of the major obstacles in the biotechnology industry. Scale-down bioreactors have been identified as valuable tools to investigate the heterogeneities observed in large-scale tanks at the laboratory scale. Additionally, computational fluid dynamics (CFD) simulations can be used to gain information about fluid flow in tanks used for production. Here, we present the rational design and comprehensive characterization of a scale-down setup, in which a flexible and modular plug-flow reactor was connected to a stirred-tank bioreactor. With the help of CFD using the realizable k-ε model, the mixing time difference between a 20 and 4000 L bioreactor was evaluated and used as scale-down criterion. CFD simulations using a shear stress transport (SST) k-ω turbulence model were used to characterize the plug-flow reactor in more detail, and the model was verified using experiments. Additionally, the model was used to simulate conditions where experiments technically could not be performed due to sensor limitations. Nevertheless, verification is difficult in this case as well. This was the first time a scale-down setup was tested on high-cell-density Escherichia coli cultivations to produce industrially relevant antigen-binding fragments (Fab). Biomass yield was reduced by 11% and specific product yield was reduced by 20% during the scale-down cultivations. Additionally, the intracellular Fab fraction was increased by using the setup. The flexibility of the introduced scale-down setup in combination with CFD simulations makes it a valuable tool for investigating scale effects at the laboratory scale. More information about the large scale is still necessary to further refine the setup and to speed up bioprocess scale-up in the future.     

Scale-down model qualification of ambr® 250 high-throughput mini-bioreactor system for two commercial-scale mAb processes

Recent advances in high-throughput (HTP) automated mini-bioreactor systems have significantly improved development timelines for early-stage biologic programs. Automated platforms such as the ambr® 250 have demonstrated the ability, using appropriate scale-down approaches, to provide reliable estimates of process performance and product quality from bench to pilot scale, but data sets comparing to large-scale commercial processes (>10,000 L) are limited. As development moves toward late stages, specifically process characterization (PC), a qualified scale-down model (SDM) of the commercial process is a regulatory requirement as part of Biologics License Application (BLA)-enabling activities. This work demonstrates the qualification of the ambr® 250 as a representative SDM for two monoclonal antibody (mAb) commercial processes at scales >10,000 L. Representative process performance and product quality associated with each mAb were achieved using appropriate scale-down approaches, and special attention was paid to pCO₂ to ensure consistent performance and product quality. Principal component analysis (PCA) and univariate equivalence testing were utilized in the qualification of the SDM, along with a statistical evaluation of process performance and product-quality attributes for comparability. The ambr® 250 can predict these two commercial-scale processes (at center-point condition) for cell-culture performance and product quality. The time savings and resource advantages to performing PC studies in a small-scale HTP system improves the potential for the biopharmaceutical industry to get products to patients more quickly.     

A systematic approach for scale-down model development and characterization of commercial cell culture processes

The objective of process characterization is to demonstrate robustness of manufacturing processes by understanding the relationship between key operating parameters and final performance. Technical information from the characterization study is important for subsequent process validation, and this has become a regulatory expectation in recent years. Since performing the study at the manufacturing scale is not practically feasible, development of scale-down models that represent the performance of the commercial process is essential to achieve reliable process characterization. In this study, we describe a systematic approach to develop a bioreactor scale-down model and to characterize a cell culture process for recombinant protein production in CHO cells. First, a scale-down model using 2-L bioreactors was developed on the basis of the 2000-L commercial scale process. Profiles of cell growth, productivity, product quality, culture environments (pH, DO, pCO2), and level of metabolites (glucose, glutamine, lactate, ammonia) were compared between the two scales to qualify the scale-down model. The key operating parameters were then characterized in single-parameter ranging studies and an interaction study using this scale-down model. Appropriate operation ranges and acceptance criteria for certain key parameters were determined to ensure the success of process validation and the process performance consistency. The process worst-case condition was also identified through the interaction study.