You are on page 1of 11

Chemical Engineering Science 61 (2006) 2939 – 2949

Fluid mixing in shaken bioreactors: Implications for scale-up predictions
from microlitre-scale microbial and mammalian cell cultures
M. Micheletti, T. Barrett, S.D. Doig, F. Baganz, M.S. Levy, J.M. Woodley, G.J. Lye ∗
Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK
Received 30 June 2005; received in revised form 4 November 2005; accepted 6 November 2005
Available online 30 January 2006

Pressures on pharmaceutical companies to speed bioprocess development have led to significant interest in small scale, parallel experimentation.
A particular focus is cell cultivation and the optimisation of protein synthesis because of the number of biological and engineering variables
involved. In this work, we briefly review the current understanding of mixing and mass transfer phenomena in shaken bioreactors with a view
to defining criteria for the scale-up of results obtained in shaken microwell systems to conventional laboratory scale. Scale-up approaches are
illustrated for two different cell cultures. The first involves an automated microscale process (1000 l) for the aerobic fermentation of E. coli
JM107:pQR706 overexpressing transketolase (TK) which is subsequently used for asymmetric carbon–carbon bond formation. The kinetics of
both the fermentation and bioconversion stages are first quantified as a function of fermentation medium composition (LB or LB-glycerol) and
shaking frequency with oxygen transfer rates being identified as rate limiting in certain cases. Successful scale-up of the microwell process (in
terms of maximum cell growth rate, biomass yield and specific TK activity) to a 1.4 l scale mechanically stirred bioreactor is then demonstrated
based on experiments performed at constant kL a values. The second process investigated involved antibody production in suspension cultures
of VPM8 hybridoma cells. Initial results suggest that experiments performed at constant mean energy dissipation rates provide a satisfactory
basis for scale translation from shaken microwells (800 l) to conical flasks (100 ml) and are indicative of results obtained in a mechanically
stirred bioreactor (3.5 l). Overall this work provides an initial insight into the engineering characterisation of shaken bioreactors and how key
parameters may be used to define suitable scale-up criteria for different cell cultures.
䉷 2005 Elsevier Ltd. All rights reserved.
Keywords: Fermentation; Cell culture; Biocatalysis; Scale-up; Automated microscale processing

1. Introduction
The pressure on pharmaceutical companies to cut bioprocess
development times and costs has led to significant interest in
small scale, parallel experimentation. A particular focus is cell
cultivation and the optimisation of protein synthesis because
of the number of biological and engineering variables involved
and their high degree of interaction. One approach has been
to miniaturise (5–100 ml) and re-design conventional mechanically stirred laboratory bioreactors (Gill et al., 2005; Kostov
et al., 2001; Lamping et al., 2003; Weuster-Botz, 2005). While
maintaining information content per fermentation the degree
of parallelisation and hence throughput is limited. The second

∗ Corresponding author. Tel.: +44 0207 679 7942; fax: +44 0207 679 0703.

E-mail address: (G.J. Lye).
0009-2509/$ - see front matter 䉷 2005 Elsevier Ltd. All rights reserved.

approach avoids the use of mechanical stirring relying instead
on shaking to promote fluid mixing and gas–liquid mass transfer. In the case of shaken microplate cultures (50–1000 l) the
application of laboratory automation technology enables thousands of cultures to be examined simultaneously.
Recently, there have been a number of studies on bioprocess
unit operations in microwell formats particularly microbial
fermentation (Duetz et al., 2000; Elmahdi et al., 2003) and
bioconversion (Doig et al., 2002; John and Heinzle, 2001). We
have also proposed the operation of automated whole bioprocess sequences in microwell formats (Lye et al., 2003) demonstrating the principle (Ferreira-Torres et al., 2005) and evaluating the use of current laboratory robots for bioprocess studies
(Nealon et al., 2005). If microwell processes are to be used to
inform process design, then a fundamental requirement is the
ability to relate microscale data to conventional laboratory and
pilot scales. In this work, we begin by briefly reviewing the

Micheletti et al. From the power number variation with the flask Reynolds number.2 . power consumption and oxygen transfer in shaken bioreactors In the past few decades shaken conical flasks. they have been characterised in terms of power consumption by Büchs et al. 1996. (2001) who calculated the modified power number. The second is antibody production in suspension cultures of VPM8 hybridoma cells. flask sizes and fill volumes. The approach is illustrated with two different cell cultures. “out of phase” conditions. rotating horizontal drum and can be expressed by Eq. was derived from an analogy to a partially filled. Characterisation of mixing.2940 M. (2000). Measurements were carried out with water for different shaking frequencies.. a new non-dimensional number. in which only a minor fraction of the liquid is actually moving along the flask wall and the power consumption is greatly reduced.1. (1) to within ±30%: Po = CRe−0. called the Phase number (Ph).94 using least-squares non-linear fitting of experimental data points. (2): ⎧ ⎡ ⎪ 2 ⎨ ds ⎢ (2N )df Ph = 1 + 3 log10 ⎣ df ⎪ 4 ⎩ 1/3 ⎞ ⎤⎫  1/3 2 2 ⎪ . In order to systematically describe the “out of phase” conditions. Mitra et al. Re was calculated using the maximum inner flask diameter df as the characteristic length scale and the constant C was found to be equal to 1. we then show how these can be used to define initial conditions for reliable scale-up of microscale cultures. 1.. for a wide range of experimental conditions. the results being described by Eq. with typical working volumes of 50–500 ml. 1998). two flow conditions were identified: “in phase” conditions in which the bulk of the liquid in the flask circulates in phase with the shaking table. (1) where Po is a modified power number (Po = P /N 3 df4 VL ). This work was later extended by Büchs et al. / Chemical Engineering Science 61 (2006) 2939 – 2949 current understanding of mixing and mass transfer phenomena in shaken bioreactors. Po . have been widely employed for cell culture process development. The first involves the production and use of a whole cell Escherichia coli JM107:pQR706 biocatalyst expressing transketolase (TK) for asymmetric C–C bond formation (Hobbs et al. Recently.

⎬ .

36 Frx Boy . a number of useful parameters have been suggested that begin to characterise the engineering environment in shaken flasks and microwells.1. As the above review has shown. the shaking frequency seems to play a crucial role on oxygen transfer and therefore on the outcome of fermentations with aerobic microorganisms having high oxygen demands.26) may exist when large flasks or high viscosity fluids are used. explaining the higher OTR measured previously. (2005).68 Sc0.1. The fermentation experiments used Luria-Bertani (LB) medium as well as a modified version of LB containing glycerol as the carbon source (10 g l−1 ). (2003) measured oxygen transfer rates (OTRmax ) in a single well of a standard round well plate (96-SRW) using the 2. Studies on the well geometry also revealed that OTRmax values in wells with a square cross-section are approximately twice those measured in round wells for the same shaking frequency and fill volume. (2003). Fermentation medium and inoculum preparation The strain used for the microbial TK bioconversion was E. dw (4) where ai is the initial specific surface area. The latter observation may be explained by the baffling effect of the corners of the square well.26 are “in phase” while “out of phase” conditions (P h < 1.1. there are few data available in the published literature on the subject. 4 VL ⎟ ⎥ ⎜ × ⎝1 − 1 − ⎠ ⎦ . shaking diameters and fill volumes. Gas–liquid mass transfer coefficients (kL a) values for various round-well diameters have also been reported by Doig et al. since . as well as the data obtained by Hermann et al. The liquid hydrodynamics was also monitored using a CCD camera and a critical frequency (Ncrit ) was identified at which the liquid height and surface area started to increase. Microbial fermentation and bioconversion process 2. (3):  dw Ncrit = . based on both dynamic gassing out measurements and on the growth kinetics of an obligate aerobe. They observed that. (3) was obtained for a single fill volume (VL = 200 l) in a 96-SRW plate. Among them. (3) 4VL ds Eq. The variation of the liquid height with N was measured at different shaking diameters and the experimental results showed good agreement with Eq. x and y are constants depending on the microplate geometry and the dimensionless numbers have their usual meaning. 2. DO2 is the oxygen diffusion coefficient. Duetz and Witholt. Due to the different flow hydrodynamics observed in 96-deep square well plates (96-DSW. for shaking speeds higher than a critical value. despite the importance of scale-up and its impact on the validation of microwell experiments. to within ±30%. c1 . The correlation was able to predict the data presented in their work. OTRmax increased exponentially. In addition. A relatively large number of studies have examined gas–liquid mass transfer in shaken systems. The following correlation was proposed to enable prediction of kL a values: kL a = c1 DO2 ai Re0. Hermann et al. Materials and methods According to their investigation all operating conditions at which P h > 1. ⎪  df ⎭ ⎛ (2) sulphite-oxidation method for a wide range of shaking frequencies (0–1000 rpm). 2001) a different relationship between Ncrit and the operating conditions can be expected. coli JM107:pQR706.

1% (v/v) sodium pyruvate and 1% (v/v) 200 mM L-glutamine in Corning 24-well ultra low attachment plates (Fig.7 vvm. The plates were left unsealed to allow efficient oxygen transfer recognising that there would be some liquid losses due to evaporation. Microwell and shake flask bioreactor operation Microwell fermentations were carried out in polypropylene 96-DSW in a contained environment (Fig.M. 1.1%. / Chemical Engineering Science 61 (2006) 2939 – 2949 2941 7 Microwell (LB) 6 Microwell (LB-glycerol) 10 mm 17 mm 40 mm Biomass [gDCW l-1] Shake flask (LB) 5 Shake flask (LB-glycerol) 4 3 2 1 0 0 8 mm 7 mm 17 mm 96-DSW 96-SRW 24-SRW Fig. suggested a maximum evaporation rate of 24 l h−1 per well but this had no significant effect over the time ranges for which max values were calculated. 2. coli JM107. Stirred bioreactor operation For laboratory scale fermentations. 2. Error bars represent one standard deviation between OD measurements obtained from different wells. The wells were covered with a Diversified Biotech Breathe-easy membrane to prevent excessive evaporation. All wells contained VL = 800 l of culture (initial seeding density of 1 × 105 viable cells ml−1 ). All shake flasks fermentations were performed in duplicate with a typical standard deviation in the calculation of the maximum specific growth rate of 3%.4 l (liquid height HL Tv ) of either LB or LB-glycerol medium. a 2 l mechanically stirred bioreactor was used. On each plate 48 wells were filled with either LB or LB-glycerol medium and incubated at 37 ◦ C at different agitation speeds. Rushton turbines (diameter D = Tv /3). (c) 24-standard round well (24-SRW). at a constant air flow rate of 0. 3). Aeration was achieved through a bar sparger. coli biocatalyst produced on each medium in each type of bioreactor were performed in 96-SRW plates (Fig. the bottom impeller was located at a clearance C = 0. (b) 96-standard round well (96-SRW). 2) using -hydroxypyruvate (HPA) and glycolaldehyde (GA) as substrates (35 mM initial concentration). TK bioconversion Bioconversions using whole cell E.235D. 2. 2005).. E. 2004) and higher TK expression levels (Hobbs. All reactions were performed in duplicate and the average standard deviation in the calculated initial rates of product formation was 0. The microplate . 1). Micheletti et al. coli JM107:pQR706 cultivated in two different media (LB or LB-glycerol) in 96-DSW plates and shake flasks.1. Schematic diagrams of the individual microwell formats used in this work: (a) 96-deep square well (96-DSW). The flasks were incubated at 300 rpm and T = 37 ◦ C for 8–12 h. coli growth kinetics were determined using a sacrificial well approach as described previously (Ferreira-Torres et al. Mammalian cell process A VPM 8 hybridoma cell line expressing IgG1 (directed against a 27 kDa light chain of ovine immunoglobulin) was used. (2005) and was used to inoculate the respective fermentations at 10% of the final working volume.15Tv from the vessel bottom while the spacing between the two impellers was C =1. Cells were grown in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum.1. The overnight culture was prepared as previously reported by Ferreira-Torres et al. Temperature was maintained constant at T = 37 ◦ C and the dissolved oxygen tension (DOT) was measured using an Ingold DOT probe. Experiments were conducted at different impeller speeds that were maintained constant throughout each fermentation. 1994) than other LB modifications. The vessel was equipped with four equally spaced vertical baffles of width B = Tv /10 and thickness tb = Tv /100 and was stirred by two 2 4 6 Time [hr] 8 10 12 Fig. The bioconversion experiments were performed as described by Hobbs (1994) and each well was analysed for HPA and L-erythrulose (product) concentration by HPLC.1. Control experiments. 2. having an internal diameter Tv = 120 mm and filled with a total liquid volume VL = 1.2. Fermentation kinetics of E. The final working volume in each well was VL = 1 ml. TK expression is constitutive hence no induction is required. located below the bottom impeller.2. The pH was not controlled in order to provide a fair comparison with corresponding small scale studies. 2.. previous studies have shown that LB-glycerol cultures obtain higher cell yields (Losen et al.3. Shake flasks fermentations were performed in 1 l shake flasks inoculated with the same overnight cultures as used in microwell experiments. using pure water. In E.4.

2 the calculated values of the specific growth rate  and Xfinal are listed in Table 1(a) and (b) for LB and LB-glycerol media. 500 ml of exponentially growing culture. 2001). coli fermentation and bioconversion kinetics The overall TK process comprises aerobic fermentation. (2005). L-lactate and glutamine were all measured using an YSI 2700 select bio analyser. The growth curves obtained in the two geometries with either LB or LB-glycerol media show similar kinetics during the exponential phase and similar final biomass yields. The vessel was unbaffled and stirred by a single three-blade segment impeller (D = 70 mm) which had a measured power number of 0.38 at Re = 17 600 (Barrett 3. Quantification of E. / Chemical Engineering Science 61 (2006) 2939 – 2949 et al. Microscale TK bioconversion kinetics using E. was incubated at T = 37 ◦ C in a 5% (v/v) CO2 atmosphere and N = 120 rpm using an orbital shaker with shaking diameter ds = 20 mm. Samples withdrawn from the shake flask and bioreactor experiments were analysed using a U-2001 UV/Vis Spectrophotometer. coli fermentation broth samples was measured in 96-SRW plates using a Tecan Genios Microplate Reader at 600 nm. Analytical techniques HPA 30 L-erythrulose y = f(x) 20 y = abx 10 0 0 40 20 60 80 100 Time [min] (a) 50 L-erythrulose concentration [mM] Biocatalyst from microwell (LB) Biocatalyst from microwell (LB-glycerol) 40 Biocatalyst from shake flask (LB) Biocatalyst from shake flask (LB-glycerol) 30 20 10 3. In all experiments the maximum loss of liquid through evaporation was less than 8%. For laboratory scale cultures. (b) comparative kinetics of L-erythrulose formation obtained using the whole cell biocatalyst produced in two different media (LB and LB-glycerol).1 vvm. Samples for analysis were withdrawn daily from duplicate flasks. were established for each process using the Genesis Workstation (Tecan). to produce the recombinant biocatalyst. The total gas flow rate was constant at 0. Ammonium concentration was determined using an indophenol blue assay as described in Barrett et al. Micheletti et al.4 g ml−1 ). Glucose..2. It was incubated in an orbital shaker at N = 120 rpm and T = 37◦ C as above. Results and discussion 0 0 (b) The optical density (OD) of the E. 2. A 250 ml plastic shake-flask with a working volume of 100 ml was also used to grow the culture. The temperature was controlled at 37 ◦ C and a mixture of O2 /N2 /CO2 was sparged into the culture medium to maintain the dissolved oxygen at 40% saturation and pH at 7. 2005).6 ng ml−1 standard of purified murine IgG. Initial fermentations profiles as indicated in Fig. Samples for analysis were withdrawn daily from at least three sacrificial wells. Automated microscale process sequences. The cell number and viability of VPM8 hybridoma cells were assessed using trypan blue exclusion and a haemocytometer. The concentration was measured against a 1000–15. Initially factors influencing the kinetics of the linked process sequence in microwell and shaken flasks are described before considering further process scale-up. respectively.Braun BIOSTAT䉸 B-DCU mechanically stirred bioreactor. The secreted mouse IgG was captured using an anti-mouse IgG (Fab specific) at a concentration of 2 g ml−1 and detected using a Antimouse IgG (Fc specific) peroxidase conjugate (1. 60 Concentration [mM] 50 40 2.3. coli fermentations . All OD measurements were performed in duplicates and the maximum standard deviation of the readings was 5%. 3. 2005). was used to inoculate a 5 l B.2942 M. followed by a whole cell bioconversion.5 l (liquid height HL 2Tv ). having an internal diameter Tv = 160 mm and filled with a total liquid volume VL = 3. Accurate determination of both the maximum growth rate (max ) and the final biomass concentration (Xfinal ) are crucial for bioprocess design purposes. 2 shows representative profiles of parallel E. HPLC was used to quantify HPA and L-erythrulose concentrations throughout the TK bioconversion as described previously (Miller. Fig. Antibody titre was quantified using a “sandwich” ELISA performed in a NUNC Maxisorp 96-SRW plate. The microscale fermentation was carried out in 96-DSW plates as this geometry is widely recognised as providing the best conditions for oxygen transfer (Duetz and Witholt. The values obtained on LB-glycerol are typical of batch E. involving the linked fermentation and bioconversion operations. 20 40 60 Time [min] 80 100 120 Fig. coli JM107:pQR706 fermentations carried out in a 96-DSW plate (N = 1000 rpm) and in a 1 l shake flask (N = 300 rpm).1. For the fermentations shown in Fig. coli JM107:pQR706: (a) example of substrate (HPA) depletion and calculation of initial rate of product (L-erythrulose) formation.

In order to characterize the liquid hydrodynamics in both microwells and shake flasks. however.0 0. (2004) obtained similar final E.83 1445 700 1000 52.06 0.5 l) 150 17 000 na 0. (2004). the maximum inner flask diameter and the impeller diameter as the characteristic length scale in the three different geometrics. Microbial and mammalian cell processes performed in 96-DSW and 24-SRW formats. (2001). The density and kinematic viscosity of water at the working temperature (T = 37 ◦ C) were used in all calculations.9 28. Micheletti et al.4 l) OTRmax (mmol l−1 h−1 ) N (rpm) Fermentation Bioconversion  (h−1 ) Biomass (g l−1 ) Initial rate (U ml−1 ) Specific activity (U g−1 DCW ) 12.8 0.1. coli biomass concentration in LB and LB-glycerol of approximately 2 and 5 g l−1 .45 1.60 6.44 1. shake flask and stirred-tank bioreactors Bioreactor geometry (a) Microbial cell process Microwell (1000 l) Shake flask (100 ml) Stirred tank (1.39 5.2 0.077c 0. c Calculated using the correlation of Linek et al.8 116.7 0. while Re is calculated and reported in Table 2(a).7 1.4 l) (b) Mammalian cell process Microwell (800 l) N (rpm) Rea 500 750 1000 700 1060 1400 300 106 800 700 1000 24 720 35 320 120 730 Ph PG /VL (kW m−3 ) kL a (s−1 ) nd nd nd nd nd nd 0.00364 nd.1 48. coli JM107:pQR706 fermentation and bioconversion process kinetics in microwell.7 0.2 0.6c nd 0.10 0.50 2383 700 1000 52.1 48.37 0.9 1.71 13 540 7674 1550 300 nd 0.072 2. respectively. For example. Losen et al. shake flask and mechanically stirred bioreactors Bioreactor geometry (a) LB medium Microwell (1000 l) Shake flask (100 ml) Stirred tank (1.17c 8. not determined. a Reynolds numbers were calculated using the well diameter. 1990). along with the engineering parameters outlined in Section 1..067 nd 0.7 0.037d 0.8 0.83 1.7 0.4 l) (b) LB-glycerol medium Microwell (1000 l) Shake flask (100 ml) Stirred tank (1. na. Calculation of the Ph number . respectively.043b Stirred tank (3.25 0.68 0. / Chemical Engineering Science 61 (2006) 2939 – 2949 2943 Table 1 Summary of linked E.041 0. b Calculated using the correlation of Büchs et al. the shaking frequency.8 0. (2005).6 2. based on the reported values of the yield coefficient Yx/c for microbial aerobes (Neidhardt et al. no study has been carried out relating Re to flow regimes in microwells.8 6.04 na na 2.09 0.8c 11.8 116.0 5. is reported in Table 2(a).45 0.06 nd 1820 nd 500 750 1000 nd. that mixing characteristics in microwells have yet to be well established and very few parameters characterising flow dynamics in such geometries have been suggested to date. It is noteworthy.55 0.9 28.M. not determined.6b 3.95 3. d Based on the CFD predictions of Barrett et al. The Reynolds number in shaken systems is commonly calculated using the microwell or maximum shake flask diameter (df = 127 mm) as the characteristic length.54 nd 2916 nd 500 750 1000 12.018 0.64 9491 6740 3631 300 nd 0.017 Shake flask (100 ml) 120 18 400 2.7 4. not applicable.37 0. Table 2 Summary of the main flow conditions and scale-up parameters for the microwell.4 1.43 0.31 0.

respectively. however. Values of the initial rates and specific activity (initial rate per unit of dry cell weight) are reported in Table 1(a) and (b) for LB and LB-glycerol.2. 3. (4). Again. Scale translation of E. An additional indication that the flow in the microwell geometry is turbulent enough to provide good mixing can be found from calculation of the critical speed. The comparable biomass and  values obtained in microwells and shake flask suggests. (4) was obtained in round wells.C. 3(b) shows L-erythrulose formation kinetics using the various whole cell biocatalyst produced from the fermentations shown previously in Fig. 4(a) and (b) for microwell fermentations using LB and LB-glycerol media. the estimated kL a value is 0. which is much lower than the agitation speed (N = 1000 rpm) employed. 2003).2. liquid fill volume (VL =1000 l) and water physical properties. respectively. a culture of JM107:pQR711 grown in shake flasks using LB medium was reported to have a specific activity of 2758 U g−1 DCW . GA was chosen as the acceptor as previous investigations on E. at which a significant increase in the gas–liquid surface area is expected (Hermann et al. / Chemical Engineering Science 61 (2006) 2939 – 2949 using Eq. then the limit towards zero of the time derivative was calculated in each case. coli microscale fermentations. In this case Eq. measured the TK activity of different E. coli JM107:pQR706 were repeated at lower shaking frequencies of N =500 and 750 rpm. (6) for which R 2 > 0. Furthermore.3.1). within 30%. that the fluid in the microwells was well mixed and the cells supplied with sufficient oxygen. Given the different bioreactor geometries and fluid dynamics at the two scales estimates of the respective oxygen mass transfer rates can be made based on predicted kL a values (Section 1.2. there is a decrease in TK specific activity at high shaking frequencies and growth rates. Based on the shaking diameter (ds =3 mm). The initial rate of product formation is a crucial parameter in describing enzyme kinetics especially the relative activities with different substrates. fermentations with E. However.06 s−1 . To facilitate this. In vitro. 3. a systematic and automatable method of accurately calculating the initial rate is needed. Ncrit . coli strains using HPA and GA under similar conditions to those used here. Eq. at least. 1994). For both media an increase in final biomass yield with frequency can be noted. To explore the significance of oxygen transfer on the process. The increase in all growth rates and biomass yields with increasing shaking frequency would suggest that oxygen transfer limitations exist at the lower frequencies such that oxygen becomes the growth-limiting nutrient.2944 M. can be used to calculate microwell kL a values. the experimental data were fitted with a mathematical curve (y = f (x)) using a regression analysis. coli TK specificity have shown it to be a rapidly converted substrate (Hobbs.. and therefore ideal for promoting oxygen transfer. 3(a). (6) f (x) = yo + a(1 − e−bx ). which is close to the value reported for LB medium in Table 1(a). 1958). show cell growth kinetics at three different shaking frequencies. with a view to defining an appropriate basis for scale-up. For the fermentation with LB-glycerol the high growth rates and biomass yields would be expected to result in cultures with a high oxygen demand. as seen for the LB-glycerol medium. 2005). Micheletti et al. (2) shows that the flow in shake flasks is “in phase”. the specific growth rate also increases with N especially in the case of LB-glycerol. frequency (N =1000 rpm). This is sufficient to show that the specific TK activity obtained with LB medium is approximately twice that obtained with LB-glycerol.. (3) gave a value of Ncrit = 30 rpm. Values of the specific activity obtained in the microwell (N = 1000 rpm) and corresponding shake flask experiments agree reasonably well.99 in all cases. Furthermore. CO2 is released and the reaction is shifted to completion and becomes irreversible (Srere et al. thus giving an accurate estimate of the initial reaction rate. During the E. is a common feature in the overexpression of recombinant proteins. Miller (2005). Fig. Given the potentially large amount of data that can be obtained from microwell experiments.1). The decrease in enzyme synthesis with increased cell growth rate and yield. Effect of shaking frequency on microscale fermentation kinetics and enzyme activity In the previous section.. Eq. Calculation of Ph in microwells under the conditions used here was not possible (a negative value inside the square root is obtained). If microscale data are to be used to inform process design then it is necessary to define more precisely the conditions under which data are collected such that it can be related to conventional laboratory scale stirred tank fermentations. when HPA is used as ketol donor. coli cells is shown in Fig. being equal to the initial rate of product formation (ri ): df (x) (5) = ri . Hermann et al. lim x→0 dx All curves were fitted using Eq. In vivo. the influence of fermentation medium composition on the performance of the linked microwell fermentation–bioconversion process (at N = 1000 rpm) was demonstrated. 2. coli TK studies can be difficult due to the widely different strains and reactions used. for each of the two media.1. A typical curve for the formation of Lerythrulose from HPA and GA by whole E. Comparison of the enzyme activity obtained in this work with previous E. Among other strains. the only available correlation in the literature. as confirmed by the calculated values for  in Table 1(a) and (b). (2003) found that OTRmax in a square- . coli cultures based on oxygen transfer considerations The previous sections have shown oxygen transfer considerations to be crucial in E. it can be assumed scaleable results can be obtained if it is ensured that both microwell and laboratory scale processes occur under none oxygen transfer limited conditions as initial results have suggested (Ferreira-Torres et al. TK catalyses a reversible step in the pentose phosphate pathway in the presence of the cofactors TPP and Mg++ ions. Good agreement was obtained between bioconversions catalyzed by whole cells grown in the same medium for each bioreactor geometry. coli fermentation the plasmid pQR706 constitutively expresses TK (E. Fig.

At the lower speed. In contrast. Both biomass growth and DOT levels were measured during the fermentations as shown in Fig. Linek et al. approximately equal to the liquid-phase mass transfer coefficient. fermentations in the 2 l bioreactor were subsequently carried out at two agitation speeds. The aforementioned value. (9): 3 1 5 N = 1000 rpm 4 3 2 1 0 0 (b) 2945 2 4 6 8 10 12 Time [hr] Fig. in the fermentation at N =1000 rpm the DOT remained constant at 100% throughout. the DOT measured at N = 700 rpm decreased after approximately 2 h.58 vs . This is done at a stirring speed of 700 rpm and an air flow rate of 1 l min−1 . coli JM107:pQR706 fermentation kinetics at different shaking speeds in 96-DSW plates: (a) LB medium.2 mol m−3 (Tromans. A typical value for oxygen solubility in aqueous systems at atmospheric pressure and T = 30 ◦ C is around 1.17 s−1 is significantly higher (Table 2(a)). Micheletti et al. 700 and 1000 rpm. CL is the oxygen concentration in the liquid phase and CL∗ is the saturation concentration of oxygen. (b) LB-glycerol medium. In order to compare the results obtained in microwells and in the stirred bioreactor under matched kL a conditions.699 0. Microscale E. respectively (Table 1(a)). The oxygen partial pressure in the mixture was taken into account and CL∗ was estimated to be 0. As expected the calculated OTRmax values for the microwell (N = 1000 rpm) and stirred bioreactor (N = 700 rpm) experiments at matched kL a values are similar. reaching a minimum value of 60%.7 and 1. estimation of the ungassed to gassed power ratio in a stirred tank can be quite difficult due to the fact that previous investigations have dealt with specific geometries and operating conditions. a is the specific gas–liquid interfacial area. 4. 4 are also shown in Table 2(a)). kL . At N = 700 rpm. Error bars represent one standard deviation between OD measurements obtained from different wells.365 + gv s . was therefore corrected using the correlation provided by Quicker et al. and at constant air flow rate (1 l min−1 ) using both LB and LB-glycerol media. (2004) obtained correlations for power input and kL a in a vessel most similar to that used here (Tv = 0. vs . kL a = 0.19 mol m−3 . The corresponding OTR during the respective microwell and stirred tank fermentations can be calculated from Eq. A value of kL a = 0. However. / Chemical Engineering Science 61 (2006) 2939 – 2949 stirred by two Rushton turbines (D = Tv /3): 7 N = 500 rpm PG = 0. respectively.079 s−1 can thus be estimated for microwell fermentations at N = 1000 rpm (kL a values calculated for the lower shaking frequencies used in Fig.141 Vs−0. is close to zero.8 g l−1 . the fact that DOT reached approximately zero after 4 h of growth may suggest oxygen . van’t Riet (1979) has reviewed a large number of investigations on gas–liquid mass transfer in stirred vessels and has correlated the results to an accuracy of 20–40%. 5(a) and (b) for LB and LB-glycerol media.0108 VL 6 N = 750 rpm N = 1000 rpm Biomass [gDCW I-1] 5 4 2 0 0 2 4 6 8 10 12 Time [hr] (a) 7 N = 500 rpm 6 N = 750 rpm Biomass [gDCW I-1] (7) (8) Using these correlations a kL a value of 0.155N 3. and hence CL . Results obtained in our laboratory in 96-DSW plates have shown that kL a values are generally 30% higher than those obtained in 96-SRW plates (results not shown). However. respectively. depending on the specific gassed power and the superficial gas velocity. The maximum OTR (OTRmax ) will occur when DOT. Values of OTRmax estimated for the microwell and mechanically stirred fermentations performed at various agitation speeds are summarised in Table 1(a) and (b) for LB and LB-glycerol media.M. the calculated kL a of 0. in the case of poorly soluble gases such as oxygen in aqueous systems. With LB medium growth kinetics obtained at N = 700 and 1000 rpm were characterised by similar values of  and maximum dry cell weights of 1. (1981). It is well known that oxygen solubility may be significantly decreased by the presence of ions and electrolytes in the fermentation media. (9) where KL is the overall mass transfer coefficient. shaped deep well is about twice that in a round well of the same diameter. A relatively large amount of information has been published on kL a measurements in mechanically stirred bioreactors. Similar observations can be made for LB-glycerol fermentations (Fig.077 s−1 is similar to the highest value obtained in microwells while at N = 1000 rpm the kL a value of 0. 5(b)). commonly used for water.29 m) OTR = KL a(CL∗ − CL ). VL   PG 0. 2000).077 s−1 is obtained as the best available approximation.

At the higher N = 1000 rpm the DOT values remained constant at 100% throughout the process. for the linked fermentation–bioconversion process. Scale-translation of hybridoma cultures based on power consumption The oxygen uptake rate of mammalian cells has been shown to be in the range 2. The maximum biomass concentrations obtained both bioreactors in LB (2. Scale translation of linked fermentation–bioconversion process Finally. Likewise. N = 1000 rpm 3 Biomass. Micheletti et al. the three biomass curves (Fig. coli JM107:pQR706 fermentation and bioconversion process kinetics at microwell (1 ml). 6(a)) exhibited similar values of max and Xfinal as summarised in Table 1(b). N = 1000 rpm Biomass.7 g l−1 ) and LB-glycerol (5.4. For the microwell (N = 1000 rpm) and mechanically 0 0 (b) 20 40 60 80 Time [min] 100 120 140 Fig. N = 1000 rpm DOT. At matched kL a values. E. The corresponding fermentations and bioconversion data.85 g l−1 ) media were also very similar confirming kL a as a useful first basis for scale translation between the two geometries. shake flask (100 ml) and stirred tank (1.M. stirred (N = 700 rpm) bioreactors. N = 700 rpm 40 DOT.10×10−12 g cell−1 h−1 whereas for bacteria . when the process was repeated with LB medium.84 and 5. For the shaken flask (N =300 rpm) no estimate of kL a is available however it is noted from Table 2(a) that the energy dissipation rate is similar. Apart from some differences in the duration of the lag phase in microwell cultures.5.37 h−1 ) were obtained in LB medium in both geometries while similar max values were obtained in LB-glycerol medium.4 l) bioreactor scales: (a) cell growth. coli JM107:pQR706 growth kinetics and dissolved oxygen tension (DOT) obtained at different agitation rates in the 2 l stirred tank: (a) LB medium. (b) LB-glycerol medium. shake flask and mechanically stirred bioreactors are shown in Fig. Scale comparison of linked E. for LB-glycerol medium. limitations begin at this point resulting in a slightly lower value of  than that obtained in the corresponding microwell experiment. fermentations were performed at matched kL a values as described previously. the performance in both media was very similar at the two scales. 3. growth kinetics and L-erythrulose formation kinetics obtained in microwell. N = 700 rpm DOT. respectively. 5. N = 700 rpm 20 1 8 10 12 50 80 4 6 Time [hr] (a) 120 2 3 0 7 3 Stirred bioreator (N = 700 rpm) 4 1 8 Shake flask (N = 300 rpm) 5 20 L-erythrulose concentration [mM] 2 0 Biomass [gDCW I-1] Microwell (N = 1000 rpm) 80 1 Microwell (N = 1000 rpm) Shake flask (N = 300 rpm) 40 Stirred bioreator (N = 700 rpm) 30 20 10 0 0 0 (b) 7 Biomass [gDCW I-1] 7 DOT [%] Biomass [gDCW I-1] 2946 2 4 6 Time [hr] 8 10 12 Fig. 6. (b) L-erythrulose formation. 3. the rates of L-erythrulose formation when the cells from the respective fermentations are used for the subsequent HPA/GA bioconversion are also similar as confirmed by the specific activity values in Table 1(b).0 and 1. 6(a) and (b). confirm the good agreement observed between microwell and mechanically stirred bioreactor experiments under matched kL a conditions. N = 700 rpm 60 DOT [%] 4 40 2 0 4 Time [hr] (a) 6 2 0 0 2 4 6 100 5 60 Biomass. Identical  values (0. / Chemical Engineering Science 61 (2006) 2939 – 2949 120 6 100 5 6 Biomass. N = 1000 rpm DOT.

Overall the similar growth profiles obtained for shake flask and microwell cultures suggest that matched power consumption is. 1997).046 and 0. shake flask and stirred-tank cultures. 1999.. 2005). 1993.. m−1 . vessel geometry. By defining experimental conditions that gave matched kL a values.0E+05 2. Although this could be the results of lactate production from nutrients other than glucose (Sureshkumar and Muthanaransan. In all three systems. s−1 . 2003). 7. initially. Hashimura et al. The experiments showed that oxygen transfer is a critical parameter in microwell cultures and therefore a critical parameter for scale-up predictions to mechanically stirred bioreactors. respectively. a high value for Ylac/glu was found. During a transient transfection process. shake flasks and also a mechanically stirred bioreactor used here for the culture of VPM8 hybridoma cells. / Chemical Engineering Science 61 (2006) 2939 – 2949 Viable cell count [cells mI-1] 1. 7(a) shows the growth kinetics of VPM8 hybridoma cells in microwell. 2947 25 20 15 10 5 0 0 20 40 60 Time [hr] (b) Fig. (2001). it suggests that cells are subjected to environmental stresses in the microwell culture which might account for the lower growth rate. Fig.0E+05 0.5 l) bioreactor scales: (a) cell growth kinetics.. (6). a good basis for translation between geometries and scales. Consequently. In the case of shaken microwell cultures both the production rate and final titre are significantly higher than those in the shaken flasks and stirred bioreactor. In addition. 1987). 2005) and compare reasonably well with previously reported data (Ryu and Lee. Notation a ai b specific gas–liquid interfacial area. Micheletti et al. the potential for microscale process sequences to quantitatively distinguish the performance of cultures grown in different media was demonstrated. Concluding remarks In this study. have considered power consumption as an initial basis for scale translation (Barrett et al.2.. 2005) as this has an impact on both mixing and mass transfer characteristics. The yield coefficients for shake flask and mechanically stirred bioreactor are similar (Barrett et al.8 ml). at least. the viable cell density reached 1. in microwells. the fermentation and bioconversion kinetics of the whole cell biocatalyst E.33 × 10−3 g cell−1 h−1 .0E+05 6. The liquid in both the shaken flask and microwell moves ‘in phase’ with the orbital motion of the shaker platform.64 W m−3 (Barrett et al. a variety of bases for scale-up have been proposed including fluid turnover. Xie et al. it was shown that microscale results could be predictive of those obtained in shaken flasks and conventional bioreactor configurations at the laboratory scale. constant in Eq. the maximum cell densities were similar. Scale comparison of VPM8 hybridoma cells cultures at microwell (0. However. is a common feature in industrial cell culture processes (Dinnis and James. as found in microwells. and generally not considered limiting with regard to cell growth (Lavery and Nienow. for studies on antibody production in suspension mammalian cell cultures. the maximum growth rate in the shaken microwell was slightly less at 0. 7(b). 4. Similarly.040 h−1 . Considering power consumption.0E+05 4.0E+06 Stirred bioreator (N = 150 rpm) 8. m−1 constant in Eq.4E+06 Shake flask (N = 120 rpm) 1. 2005).46. Our current work is applying these methods to the analysis of larger libraries of evolved biocatalysts and mammalian cells. Varley and Birch. This increase in specific antibody production at retarded growth rates. mol l−1 specific static gas–liquid interfacial area. constant power consumption has been identified for the initial scale comparison of mammalian cell cultures. compared to 22 and 16 mg l−1 in the shaken flask and stirred tank. impeller tip speed and aeration rate (Chisti. and others.1 × 106 cells ml−1 . Error bars represent one standard deviation between measurements obtained from different wells. shake flask (100 ml) and stirred tank (3. 2005. respectively. The oxygen mass transfer coefficients required in mammalian cell cultures are therefore significantly lower. Maximum growth rates in the shake flask and stirred tank were found to be almost identical at 0.0E+00 0 20 (a) 40 60 Time [hr] 80 100 80 100 45 Microwell (N = 120 rpm) 40 Shake flask (N = 120 rpm) 35 Stirred bioreator (N = 150 rpm) 30 lgG1 [mg I-1] this value is in the region of 0.1.048 h−1 . After approximately 3 days of culture. Table 2(b) shows parameters describing the flow characteristics of shaken microwells.2E+06 Microwell (N = 120 rpm) 1. We. they found two-fold greater recombinant protein expression by HEK 293 cells in agitated 12-well plates compared to a 3 l stirred bioreactor. (6). The corresponding antibody production kinetics are shown in Fig. This analysis suggests than power consumption can be used as an engineering basis to compare the results of different shaken bioreactor geometries. coli JM107:pQR706 were determined using different shaking frequencies. (b) antibody production.M.. 1990). This was confirmed by visual observation. Similar results have been reported by Girard et al. The final titre obtained in microwells was 37 mg l−1 .0. However. similar values (∼ 40 W m−3 ) were found for shaken flask and microwell while the prediction for the mechanically stirred bioreactor is a factor of 10 lower at 3.

W modified power number. Cell culture scale-down model development through impeller agitation rate optimization.. Ferreira-Torres.D. m impeller diameter. 191–196. 2001. m s−1 liquid volume. Zoels. Levy. Financial support from the 12 industrial partners supporting the BiCE programme is also acknowledged..... References Barrett.R.. London.J. Chemical Engineering Science 60. Micheletti et al. Peacock. Animal cell culture in stirred bioreactors: observations on scale-up. mol l−1 s−1 Reynolds number (=N d 2 /). Ph. L. Biotechnology Bioengineering 68. dimensionless time. Milbradt. W gassed power consumption. F. D.. The use of microscale processing technologies for quantification of biocatalytic Baeyer-Villiger oxidation kinetics. M. / Chemical Engineering Science 61 (2006) 2939 – 2949 baffle width. Wurm. dimensionless initial rate of product formation.. Hermann. Biochemical Engineering Journal. Pickering. R. W. Power consumption measurement in unbaffled flasks at low liquid viscosity. Büchs. R. Small-scale bioreactor system for process development and optimization. Sugden. F. F.D. dimensionless constant in Eq. m shaken microwell diameter. Biochemical Engineering Journal 16. 1996. 2000. N m−1 Acknowledgements The authors would like to thank the UK Engineering and Physical Sciences Research Council (EPSRC) for support of the multidisciplinary Biocatalysis Integrated with Chemistry and Engineering (BiCE) programme (GR/S62505/01). mol l−1 oxygen concentration uptake by the cells.. m oxygen diffusion coefficient...R. Lye. Biochemical Engineering XIV.. . mol l−1 shaken flask diameter. Hashimura. Out-of-phase conditions. dimensionless constant in Eq..D. Engineering characterisation of hybridoma cells in shaken microwell plates: application to high-throughput process development.M.. Y. Lye. Witholt.. M. G. B. m s−1 impeller rotational speed. 2005.. submitted for publication..K. Applied and Environmental Microbiology 66. J.. 1993. S. m dynamic viscosity. 2005.. m3 wetting tension. Li. m shaken diameter.. T. 117–119.M. Characterization of gas–liquid mass transfer phenomena in microtiter plates. m bond number (=gd 2w /W ). Lilly. Girard. Dinnis. J.. B. 2741–2750. Witholt. dimensionless gravitational acceleration.. erythraea CA340: influence on biomass growth kinetics and erythromycin biosynthesis. S.J.A. shaking frequency. s baffle thickness. James. dimensionless Schmidt number (=/DO2 ). 299–310. B. Duetz.K. J..J. 2005. M. G. 589–593. 42–49. dimensionless oxygen concentration in the liquid phase. Büchs. Harrison Hot Spring. Büchs. Dixon.A. U.S... G.. W. B. 2001. pH control in microwell fermentations of S. constant in Eq... Harrop. H. 2000... (4)..A. T. N m−1 constant in Eq.J. Maier. G.. M. M.C. 178–186. Design and instrumentation of a novel miniature bioreactor system for high throughput fermentation optimisation... Journal of Biotechnology 45.. Lye. Baganz. growth. m vessel internal diameter. I.. mol l−1 saturation concentration of oxygen in the liquid phase.. Bioprocess Engineering 9. Biotechnology Bioengineering 81. The authors would also like to thank the UK Joint Infrastructure Fund (JIF). J. D. Modelling surface aeration rates in shaken microtitre plates using dimensionless groups. BC. kg m−3 liquid surface tension. F. 2005. Hermann. Effectiveness of orbital shaking for the aeration of suspended cultures in square-deepwell microtiter plates. S. mol l−1 s−1 power consumption. Micheletti.M. (4). S.. R. Woodley. D. K.J.. Zhang. Tsao. 10–14 July 2005.M. Support from the Biotechnology and Biological Sciences Research Council is also acknowledged in the form of a studentship for TB. Lehmann. mol l−1 Greek letters C  max   spacing between impellers. 2002. Biochemical Engineering Journal 7. Baganz. 2001.. Biotechnology Bioengineering 80. P. 135–141.R. Gill... Engineering mammalian cell factories for improved recombinant monoclonal antibody production: lessons from nature? Biotechnology Bioengineering 91. C. Baganz.. Methods for intense aeration. s−1 maximum growth rate. storage and replication of bacterial strains in microtiter plates.J. Y. m−1 off-bottom clearance. Doig.. 2005. Canada.C. J. G. 2003. Chisti. K.. m2 s−1 froude number (=ds (2N )2 /2g).. submitted for publication. Rüedi. Büchs. R. ◦ C superficial gas velocity. Doig. 2641–2646. Lye. N. specific growth rate.. C. Woodley.. The product and use of transketolase as a catalyst for carbon–carbon bond formation. Duetz. O’Connor. Enzyme-catalysed carbon–carbon bond formation: large-scale production of Escherichia coli transketolase. m. a hitherto unknown phenomenon in shaking bioreactors. 83–93. S. m s−2 liquid height.. (4).. Milbradt. m liquid-film mass transfer coefficient. 1994. G. mol l−1 constant in Eq. M. Mitra. Chauhan. Lye.. Pickering. M. 2005. Lotter. J.. Thesis. Power consumption in shaking flasks on rotary shaking machines: I. dimensionless biomass concentration.. (6). Hobbs.. s−1 oxygen transfer rate.C. Jordan. Microscale process evaluation of recombinant biocatalyst libraries: application to BayerVilliger monooxygenase catalysed lactone synthesis. F. Biosystems Bioengineering 28. m temperature. m s−1 overall mass transfer coefficient. the Science Research Investment Fund (SRIF) and the Gatsby Charitable Foundation for funds to establish the UCL Centre for Micro Biochemical Engineering. Biochemical Engineering Journal 7. G. 173–179. Hobbs. (1).. Elmahdi. 113–115.P. Biotechnology Bioengineering. 2003. Biochemical Engineering Journal 7. Lye. UK. 180–189.. G. University College London.2948 B Bo c1 C CL CL∗ CO2 df ds dw D DO2 Fr g HL kL KL N OTR P PG Po ri Re Sc t tb Tv T vs VL W x X y yo M.. kg m−1 s−1 ...D. Lee.R. C. s−1 liquid density. M. Appleton..

. Biotechnology Bioengineering 37. Design of a prototype miniature bioreactor for high throughput automated bioprocessing.M.D. Large-scale propagation of a replication-defective adenovirus vector in stirred-tank bioreactor PER.R. W.. 747–758. 2001.J. Escherichia coli transketolase-catalyzed carbon–carbon bond formation: biotransformation characterization for reactor evaluation and selection. Preparation of substrates and enzymes. a carbon–carbon bond forming enzyme. A. MA. E. Biotechnology Bioengineering 23... M.A.D. Heinzle. Pikus. 2001... Bioprocess Engineering 16. J. 2005. A. O.. J.. Chemical Engineering Science 58. Lamping. Muthanaransan. 177–205.A. Gas–liquid mass transfer coefficient in stirred tanks interpreted through models of idealized eddy structure of turbulence in the bubble vicinity. Biotechnology Bioengineering 30.. Leung.. Enzyme and Microbiol Technology 22. M. Quicker. 45–52. J. UK. 29–37.W. 1981.. 2005. Aunins..M. T. 2004. Biotechnology Progress 21.J. 1979. M. J. Y. F. Büchs.D. D. Lowcost microbioreactor for high-throughput bioprocessing. J.. M.. 1511–1517. 2003. Modeling oxygen solubility in water and electrolyte solutions.T. M.S. W.. Zhou.. Schumpe.A. Birch.. Weuster-Botz.. P. 346–352. T... Sureshkumar.M. Comparison of measured and calculated oxygen solubilities in fermentation media... A. Effect of oxygen limitation and medium composition on Escherichia coli fermentation in shake-flask cultures.. 1987... Willson. Parallel reactor systems for bioprocess development. H.. Woodley. B. Deckwer. 1062–1068. Neidhardt.. Metallo. 1997.. G.. The influence of temperature on a mouse-mouse hybridoma growth and monoclonal antibody production. 1990. London. 1990. Miller. Industrial Engineering Chemistry Research 39. 283–291. Nienow. Mitra... T. 292–295.. Influence of hyperosmolar basal media on hybridoma cell growth and antibody production.... I. Kordac.C. R. C. Reactor design for large scale suspension animal cell culture. / Chemical Engineering Science 61 (2006) 2939 – 2949 John... 1998. Trends in Biotechnology 21. Lavery.R. L.. Tromans. Xie. Varley.C. Schaechter... 2003. University College London. Shamlou. 357–364. Biotechnology Progress 20. Oxygen transfer in animal cell culture. Lye. Advances in Biochemical Engineering and Biotechnology 92. 2003. E. Cooper. Pickering.. J.. Srere. Quantitative screening method for hydrolases in microplates using pH indicators: determination of kinetic parameters by dynamic pH monitoring... Racker. 620–627.. 368–373.. 2000.... G. Biotechnology Bioengineering 72. Accelerated design of bioconversion processes using automated microscale processing techniques..S.G. 1999. N.. G. 2005..K.L. Clayton. V. G. B. J. J. Linek.. Peltier. G.. König. Warren. 64–70. Ph. Dalby. Physiology of the Bacterial Cell. . 125–143. B. J.K. Directed evolution of transketolase. M. Ryu. L. S. K. Rao.C6 cell culture under sparging conditions. Yancy. Thesis. 105–310. P. Lee. Harms. J. L..J. S..M.J..J.. Industrial Engineering Process Design and Development 18.. M. Pohl. Zhong. F. Losen.. Zhang. Biotechnology Bioengineering 72. Micheletti et al. Allen. Woodley. P. Nealon. Randers-Eichhorn. K. M. Biotechnology Bioengineering 83. R. D. Kostov.. A. Archives of Biochemistry and Biophysics 295–305. Titchener-Hooker... Review of measuring methods and results in nonviscous gas–liquid mass transfer in stirred vessels. Sunderland.. Baganz.A. Use of operating windows 2949 in the assessment of integrated robotic systems for the measurement of bioprocess kinetics. Tabachnick. Pilbrough. 1958. G. O’Kennedy.. J.E. 635–650. 2004. Chemical Engineering and Processing 43. M. Sinauer Associates Inc. Moucha. P. J. Lye. Ingraham. P. Shamlou. Fujasová. W. 805–812. R. van’t Riet.-D. Cytotechnology 29. Lilly...R. The oxidative pentose phosphate cycle. Frölich..