Chemical Engineering Science 61 (2006) 2939 2949

www.elsevier.com/locate/ces

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

Abstract
Pressures on pharmaceutical companies to speed bioprocess development have led to signicant 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 briey review the current understanding of mixing and mass transfer phenomena in shaken bioreactors with a view
to dening 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 rst 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 carboncarbon bond formation. The kinetics of
both the fermentation and bioconversion stages are rst quantied as a function of fermentation medium composition (LB or LB-glycerol) and
shaking frequency with oxygen transfer rates being identied as rate limiting in certain cases. Successful scale-up of the microwell process (in
terms of maximum cell growth rate, biomass yield and specic 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 asks (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 dene suitable scale-up criteria for different cell cultures.
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 signicant 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 (5100 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.lye@ucl.ac.uk (G.J. Lye).

0009-2509/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ces.2005.11.028

approach avoids the use of mechanical stirring relying instead

on shaking to promote uid mixing and gasliquid mass transfer. In the case of shaken microplate cultures (501000
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 briey reviewing the

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M. Micheletti et al. / Chemical Engineering Science 61 (2006) 2939 2949

current understanding of mixing and mass transfer phenomena

in shaken bioreactors; we then show how these can be used
to dene initial conditions for reliable scale-up of microscale
cultures. The approach is illustrated with two different cell cultures. The rst involves the production and use of a whole cell
Escherichia coli JM107:pQR706 biocatalyst expressing transketolase (TK) for asymmetric CC bond formation (Hobbs
et al., 1996; Mitra et al., 1998). The second is antibody production in suspension cultures of VPM8 hybridoma cells.
1.1. Characterisation of mixing, power consumption and
oxygen transfer in shaken bioreactors
In the past few decades shaken conical asks, with typical
working volumes of 50500 ml, have been widely employed
for cell culture process development. Recently, they have been
characterised in terms of power consumption by Bchs et al.
(2000). Measurements were carried out with water for different
shaking frequencies, ask sizes and ll volumes, the results
being described by Eq. (1) to within 30%:
Po = CRe0.2 ,

(1)

where Po is a modied power number (Po = P /
N 3 df4 VL ),

Re was calculated using the maximum inner ask diameter
df as the characteristic length scale and the constant C was
found to be equal to 1.94 using least-squares non-linear tting
of experimental data points. This work was later extended by
Bchs et al. (2001) who calculated the modied power number, Po , for a wide range of experimental conditions. From the
power number variation with the ask Reynolds number, two
ow conditions were identied: in phase conditions in which
the bulk of the liquid in the ask circulates in phase with the
shaking table; out of phase conditions, in which only a minor fraction of the liquid is actually moving along the ask
wall and the power consumption is greatly reduced. In order
to systematically describe the out of phase conditions, a new
non-dimensional number, called the Phase number (Ph), was
derived from an analogy to a partially lled, rotating horizontal
drum and can be expressed by Eq. (2):

ds

(2N )df
Ph =
1 + 3 log10
df
4

1/3


 1/3 2 2



4 VL

1 1
.

 df

(2)

sulphite-oxidation method for a wide range of shaking frequencies (01000 rpm), shaking diameters and ll volumes. They
observed that, for shaking speeds higher than a critical value,
OTRmax increased exponentially. 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 ll volume. The
latter observation may be explained by the bafing effect of
the corners of the square well. The liquid hydrodynamics was
also monitored using a CCD camera and a critical frequency
(Ncrit ) was identied at which the liquid height and surface
area started to increase, explaining the higher OTR measured
previously. The variation of the liquid height with N was
measured at different shaking diameters and the experimental
results showed good agreement with Eq. (3):

dw
Ncrit =
.
(3)
4VL
ds
Eq. (3) was obtained for a single ll volume (VL = 200
l)
in a 96-SRW plate. Due to the different ow hydrodynamics
observed in 96-deep square well plates (96-DSW, Duetz and
Witholt, 2001) a different relationship between Ncrit and the
operating conditions can be expected. Gasliquid mass transfer coefcients (kL a) values for various round-well diameters
have also been reported by Doig et al. (2005), based on both
dynamic gassing out measurements and on the growth kinetics
of an obligate aerobe. The following correlation was proposed
to enable prediction of kL a values:
kL a = c1

DO2
ai Re0.68 Sc0.36 Frx Boy ,
dw

(4)

where ai is the initial specic surface area, DO2 is the oxygen

diffusion coefcient, c1 , x and y are constants depending on
the microplate geometry and the dimensionless numbers have
their usual meaning. The correlation was able to predict the
data presented in their work, as well as the data obtained by
Hermann et al. (2003), to within 30%.
As the above review has shown, a number of useful parameters have been suggested that begin to characterise the engineering environment in shaken asks and microwells. Among
them, 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. In
addition, despite the importance of scale-up and its impact on
the validation of microwell experiments, there are few data
available in the published literature on the subject.
2. Materials and methods

According to their investigation all operating conditions at

which P h > 1.26 are in phase while out of phase conditions
(P h < 1.26) may exist when large asks or high viscosity uids
are used.
A relatively large number of studies have examined
gasliquid mass transfer in shaken systems. Hermann et al.
(2003) measured oxygen transfer rates (OTRmax ) in a single well of a standard round well plate (96-SRW) using the

2.1. Microbial fermentation and bioconversion process

2.1.1. Fermentation medium and inoculum preparation
The strain used for the microbial TK bioconversion was
E. coli JM107:pQR706. The fermentation experiments used
Luria-Bertani (LB) medium as well as a modied version of
LB containing glycerol as the carbon source (10 g l1 ), since

M. Micheletti et al. / Chemical Engineering Science 61 (2006) 2939 2949

2941

7
Microwell (LB)

Microwell (LB-glycerol)

10 mm

17 mm

40 mm

Biomass [gDCW l-1]

Shake flask (LB)

Shake flask (LB-glycerol)

4
3
2
1
0
0

8 mm

7 mm
17 mm

96-DSW

96-SRW

24-SRW

Fig. 1. Schematic diagrams of the individual microwell formats used in

this work: (a) 96-deep square well (96-DSW); (b) 96-standard round well
(96-SRW); (c) 24-standard round well (24-SRW).

previous studies have shown that LB-glycerol cultures obtain higher cell yields (Losen et al., 2004) and higher TK
expression levels (Hobbs, 1994) than other LB modications.
The overnight culture was prepared as previously reported by
Ferreira-Torres et al. (2005) and was used to inoculate the
respective fermentations at 10% of the nal working volume.
2.1.2. Microwell and shake ask bioreactor operation
Microwell fermentations were carried out in polypropylene
96-DSW in a contained environment (Fig. 1). On each plate
48 wells were lled with either LB or LB-glycerol medium
and incubated at 37 C at different agitation speeds. The nal
working volume in each well was VL = 1 ml. The plates were
left unsealed to allow efcient oxygen transfer recognising that
there would be some liquid losses due to evaporation. Control
experiments, using pure water, suggested a maximum evaporation rate of 24
l h1 per well but this had no signicant effect
over the time ranges for which max values were calculated. E.
coli growth kinetics were determined using a sacricial well
approach as described previously (Ferreira-Torres et al., 2005).
Shake asks fermentations were performed in 1 l shake asks
inoculated with the same overnight cultures as used in microwell experiments. The asks were incubated at 300 rpm and
T = 37 C for 812 h. All shake asks fermentations were performed in duplicate with a typical standard deviation in the
calculation of the maximum specic growth rate of 3%.
2.1.3. Stirred bioreactor operation
For laboratory scale fermentations, a 2 l mechanically stirred
bioreactor was used, having an internal diameter Tv = 120 mm
and lled with a total liquid volume VL = 1.4 l (liquid height
HL
Tv ) of either LB or LB-glycerol medium. The vessel was
equipped with four equally spaced vertical bafes of width
B = Tv /10 and thickness tb = Tv /100 and was stirred by two

6
Time [hr]

10

12

Fig. 2. Fermentation kinetics of E. coli JM107:pQR706 cultivated in two

different media (LB or LB-glycerol) in 96-DSW plates and shake asks. Error
bars represent one standard deviation between OD measurements obtained
from different wells.

Rushton turbines (diameter D = Tv /3); the bottom impeller

was located at a clearance C = 0.15Tv from the vessel bottom
while the spacing between the two impellers was C =1.235D.
Experiments were conducted at different impeller speeds that
were maintained constant throughout each fermentation. Aeration was achieved through a bar sparger, located below the
bottom impeller, at a constant air ow rate of 0.7 vvm. The pH
was not controlled in order to provide a fair comparison with
corresponding small scale studies. Temperature was maintained
constant at T = 37 C and the dissolved oxygen tension (DOT)
was measured using an Ingold DOT probe.
2.1.4. TK bioconversion
Bioconversions using whole cell E. coli biocatalyst produced
on each medium in each type of bioreactor were performed in
96-SRW plates (Fig. 2) using -hydroxypyruvate (HPA) and
glycolaldehyde (GA) as substrates (35 mM initial concentration). In E. coli JM107, TK expression is constitutive hence
no induction is required. The bioconversion experiments were
performed as described by Hobbs (1994) and each well was
analysed for HPA and L-erythrulose (product) concentration by
HPLC. All reactions were performed in duplicate and the average standard deviation in the calculated initial rates of product
formation was 0.1%.
2.2. Mammalian cell process
A VPM 8 hybridoma cell line expressing IgG1 (directed
against a 27 kDa light chain of ovine immunoglobulin) was
used. Cells were grown in RPMI-1640 medium supplemented
with 10% (v/v) fetal bovine serum, 1% (v/v) sodium pyruvate
and 1% (v/v) 200 mM L-glutamine in Corning 24-well ultra low
attachment plates (Fig. 3). The wells were covered with a Diversied Biotech Breathe-easy membrane to prevent excessive
evaporation. All wells contained VL = 800
l of culture (initial
seeding density of 1 105 viable cells ml1 ). The microplate

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M. Micheletti et al. / Chemical Engineering Science 61 (2006) 2939 2949

et al., 2005). 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
7.2. The total gas ow rate was constant at 0.1 vvm.

60

Concentration [mM]

50
40

2.3. 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. Results and discussion

0
0
(b)

The optical density (OD) of the E. coli fermentation broth

samples was measured in 96-SRW plates using a Tecan Genios Microplate Reader at 600 nm. Samples withdrawn from
the shake ask and bioreactor experiments were analysed using
a U-2001 UV/Vis Spectrophotometer. All OD measurements
were performed in duplicates and the maximum standard deviation of the readings was 5%. HPLC was used to quantify
HPA and L-erythrulose concentrations throughout the TK bioconversion as described previously (Miller, 2005).
The cell number and viability of VPM8 hybridoma cells
were assessed using trypan blue exclusion and a haemocytometer. Antibody titre was quantied using a sandwich ELISA
performed in a NUNC Maxisorp 96-SRW plate. The secreted
mouse IgG was captured using an anti-mouse IgG (Fab specic) at a concentration of 2
g ml1 and detected using a Antimouse IgG (Fc specic) peroxidase conjugate (1.4
g ml1 ).
The concentration was measured against a 100015.6 ng ml1
standard of puried murine IgG. Glucose, L-lactate and glutamine were all measured using an YSI 2700 select bio analyser.
Ammonium concentration was determined using an indophenol
blue assay as described in Barrett et al. (2005).

20

40

60
Time [min]

80

100

120

Fig. 3. Microscale TK bioconversion kinetics using E. coli JM107:pQR706:

(a) example of substrate (HPA) depletion and calculation of initial rate of
product (L-erythrulose) formation; (b) comparative kinetics of L-erythrulose
formation obtained using the whole cell biocatalyst produced in two different
media (LB and LB-glycerol). Initial fermentations proles as indicated in
Fig. 2.

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 for analysis were withdrawn daily from
at least three sacricial wells. In all experiments the maximum
loss of liquid through evaporation was less than 8%.
A 250 ml plastic shake-ask with a working volume of 100 ml
was also used to grow the culture. It was incubated in an orbital shaker at N = 120 rpm and T = 37 C as above. Samples
for analysis were withdrawn daily from duplicate asks. For
laboratory scale cultures, 500 ml of exponentially growing culture, was used to inoculate a 5 l B.Braun BIOSTAT B-DCU
mechanically stirred bioreactor, having an internal diameter
Tv = 160 mm and lled with a total liquid volume VL = 3.5 l
(liquid height HL
2Tv ). The vessel was unbafed 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

3.1. Quantication of E. coli fermentation and bioconversion

kinetics
The overall TK process comprises aerobic fermentation, to
produce the recombinant biocatalyst, followed by a whole cell
bioconversion. Automated microscale process sequences, involving the linked fermentation and bioconversion operations,
were established for each process using the Genesis Workstation (Tecan). Initially factors inuencing the kinetics of the
linked process sequence in microwell and shaken asks are described before considering further process scale-up. 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, 2001). Fig. 2 shows
representative proles of parallel E. coli JM107:pQR706 fermentations carried out in a 96-DSW plate (N = 1000 rpm) and
in a 1 l shake ask (N = 300 rpm). The growth curves obtained
in the two geometries with either LB or LB-glycerol media
show similar kinetics during the exponential phase and similar
nal biomass yields. Accurate determination of both the maximum growth rate (max ) and the nal biomass concentration
(Xnal ) are crucial for bioprocess design purposes. For the fermentations shown in Fig. 2 the calculated values of the specic growth rate  and Xnal are listed in Table 1(a) and (b)
for LB and LB-glycerol media, respectively. The values obtained on LB-glycerol are typical of batch E. coli fermentations

M. Micheletti et al. / Chemical Engineering Science 61 (2006) 2939 2949

2943

Table 1
Summary of linked E. coli JM107:pQR706 fermentation and bioconversion process kinetics in microwell, shake ask and mechanically stirred bioreactors
Bioreactor geometry

(a) LB medium
Microwell (1000
l)

Shake ask (100 ml)

Stirred tank (1.4 l)
(b) LB-glycerol medium
Microwell (1000
l)

Shake ask (100 ml)

Stirred tank (1.4 l)

OTRmax (mmol l1 h1 )

N (rpm)

Fermentation

Bioconversion

(h1 )

Biomass
(g l1 )

Initial rate
(U ml1 )

Specic activity
(U g1
DCW )

12.9
28.1
48.7

0.10
0.25
0.37

0.9
1.6
2.0

0.83
1.09
0.64

9491
6740
3631

300

nd

0.44

1.8

0.50

2383

700
1000

52.8
116.7

0.37
0.45

1.7
1.8

0.54
nd

2916
nd

500
750
1000

12.9
28.1
48.7

0.31
0.45
0.55

0.7
4.0
5.8

0.95
3.06
0.71

13 540
7674
1550

300

nd

0.60

6.2

0.83

1445

700
1000

52.8
116.7

0.43
0.39

5.8
6.4

1.06
nd

1820
nd

500
750
1000

nd, not determined.

Table 2
Summary of the main ow conditions and scale-up parameters for the microwell, shake ask and stirred-tank bioreactors
Bioreactor geometry

(a) Microbial cell process

Microwell (1000
l)

Shake ask (100 ml)

Stirred tank (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 m3 )

kL a
(s1 )

nd
nd
nd

nd
nd
nd

0.018
0.041
0.072

2.04
na
na

2.6b
3.8c
11.6c

nd
0.077c
0.17c

8.2

0.037d

0.067
nd
0.017

Shake ask (100 ml)

120

18 400

2.68

0.043b

Stirred tank (3.5 l)

150

17 000

na

0.00364

nd, not determined; na, not applicable.

Microbial and mammalian cell processes performed in 96-DSW and 24-SRW formats.
a Reynolds numbers were calculated using the well diameter, the maximum inner ask diameter and the impeller diameter as the characteristic length scale
in the three different geometrics, respectively.
b Calculated using the correlation of Bchs et al. (2001).
c Calculated using the correlation of Linek et al. (2004).
d Based on the CFD predictions of Barrett et al. (2005).

based on the reported values of the yield coefcient Yx/c for

microbial aerobes (Neidhardt et al., 1990). Losen et al. (2004)
obtained similar nal E. coli biomass concentration in LB and
LB-glycerol of approximately 2 and 5 g l1 , respectively.
In order to characterize the liquid hydrodynamics in both
microwells and shake asks, the shaking frequency, along
with the engineering parameters outlined in Section 1.1, is
reported in Table 2(a). The density and kinematic viscosity of
water at the working temperature (T = 37 C) were used in all

calculations. The Reynolds number in shaken systems is commonly calculated using the microwell or maximum shake ask
diameter (df = 127 mm) as the characteristic length. It is
noteworthy, however, that mixing characteristics in microwells have yet to be well established and very few parameters
characterising ow dynamics in such geometries have been
suggested to date. For example, while Re is calculated and reported in Table 2(a), no study has been carried out relating Re
to ow regimes in microwells. Calculation of the Ph number

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M. Micheletti et al. / Chemical Engineering Science 61 (2006) 2939 2949

using Eq. (2) shows that the ow in shake asks is in phase,

and therefore ideal for promoting oxygen transfer. Calculation
of Ph in microwells under the conditions used here was not
possible (a negative value inside the square root is obtained).
The comparable biomass and  values obtained in microwells
and shake ask suggests, at least, that the uid in the microwells was well mixed and the cells supplied with sufcient
oxygen. An additional indication that the ow in the microwell
geometry is turbulent enough to provide good mixing can be
found from calculation of the critical speed, Ncrit , at which a
signicant increase in the gasliquid surface area is expected
(Hermann et al., 2003). In this case Eq. (3) gave a value of
Ncrit = 30 rpm, which is much lower than the agitation speed
(N = 1000 rpm) employed.
During the E. coli fermentation the plasmid pQR706 constitutively expresses TK (E.C.2.2.1.1). In vivo, TK catalyses a reversible step in the pentose phosphate pathway in the presence
of the cofactors TPP and Mg++ ions. In vitro, when HPA is
used as ketol donor, CO2 is released and the reaction is shifted
to completion and becomes irreversible (Srere et al., 1958).
GA was chosen as the acceptor as previous investigations on
E. coli TK specicity have shown it to be a rapidly converted
substrate (Hobbs, 1994). A typical curve for the formation of Lerythrulose from HPA and GA by whole E. coli cells is shown
in Fig. 3(a). The initial rate of product formation is a crucial
parameter in describing enzyme kinetics especially the relative
activities with different substrates. Given the potentially large
amount of data that can be obtained from microwell experiments, a systematic and automatable method of accurately calculating the initial rate is needed. To facilitate this, the experimental data were tted with a mathematical curve (y = f (x))
using a regression analysis, then the limit towards zero of the
time derivative was calculated in each case, being equal to the
initial rate of product formation (ri ):
df (x)
(5)
= ri .
lim
x0
dx
All curves were tted using Eq. (6) for which R 2 > 0.99 in all
cases, thus giving an accurate estimate of the initial reaction
rate.
(6)
f (x) = yo + a(1 ebx ).
Fig. 3(b) shows L-erythrulose formation kinetics using the
various whole cell biocatalyst produced from the fermentations
shown previously in Fig. 2. Good agreement was obtained between bioconversions catalyzed by whole cells grown in the
same medium for each bioreactor geometry. Values of the initial
rates and specic activity (initial rate per unit of dry cell weight)
are reported in Table 1(a) and (b) for LB and LB-glycerol, respectively. Values of the specic activity obtained in the microwell (N = 1000 rpm) and corresponding shake ask experiments agree reasonably well, within 30%, for each of the two
media. This is sufcient to show that the specic TK activity
obtained with LB medium is approximately twice that obtained
with LB-glycerol. The decrease in enzyme synthesis with increased cell growth rate and yield, as seen for the LB-glycerol
medium, is a common feature in the overexpression of recombinant proteins. Comparison of the enzyme activity obtained

in this work with previous E. coli TK studies can be difcult

due to the widely different strains and reactions used. Miller
(2005), however, measured the TK activity of different E. coli
strains using HPA and GA under similar conditions to those
used here. Among other strains, a culture of JM107:pQR711
grown in shake asks using LB medium was reported to have
a specic activity of 2758 U g1
DCW , which is close to the value
reported for LB medium in Table 1(a).
3.2. Effect of shaking frequency on microscale fermentation
kinetics and enzyme activity
In the previous section, the inuence of fermentation
medium composition on the performance of the linked microwell fermentationbioconversion process (at N = 1000 rpm) was
demonstrated. 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. To explore the signicance of oxygen transfer on the process, with a view to dening
an appropriate basis for scale-up, fermentations with E. coli
JM107:pQR706 were repeated at lower shaking frequencies of
N =500 and 750 rpm. Fig. 4(a) and (b) for microwell fermentations using LB and LB-glycerol media, respectively, show cell
growth kinetics at three different shaking frequencies. For both
media an increase in nal biomass yield with frequency can be
noted. Furthermore, as conrmed by the calculated values for
 in Table 1(a) and (b), the specic growth rate also increases
with N especially in the case of LB-glycerol. Again, there is
a decrease in TK specic activity at high shaking frequencies
and growth rates. 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.
3.3. Scale translation of E. coli cultures based on oxygen
transfer considerations
The previous sections have shown oxygen transfer considerations to be crucial in E. coli microscale fermentations. If microscale data are to be used to inform process design then it is
necessary to dene more precisely the conditions under which
data are collected such that it can be related to conventional
laboratory scale stirred tank fermentations. Given the different
bioreactor geometries and uid dynamics at the two scales estimates of the respective oxygen mass transfer rates can be made
based on predicted kL a values (Section 1.1). Furthermore, 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., 2005).
Eq. (4), the only available correlation in the literature, can be
used to calculate microwell kL a values. Based on the shaking
diameter (ds =3 mm), frequency (N =1000 rpm), liquid ll volume (VL =1000
l) and water physical properties, the estimated
kL a value is 0.06 s1 . However, Eq. (4) was obtained in round
wells. Hermann et al. (2003) found that OTRmax in a square-

M. Micheletti et al. / Chemical Engineering Science 61 (2006) 2939 2949

stirred by two Rushton turbines (D = Tv /3):

7
N = 500 rpm

PG
= 0.155N 3.141 Vs0.365 +
gv s ,
VL


PG 0.699 0.58
vs .
kL a = 0.0108
VL

6
N = 750 rpm
N = 1000 rpm

Biomass [gDCW I-1]

5
4

0
0

10

12

Time [hr]

(a)
7
N = 500 rpm

N = 750 rpm

Biomass [gDCW I-1]

(7)

(8)

Using these correlations a kL a value of 0.077 s1 is obtained

as the best available approximation. This is done at a stirring
speed of 700 rpm and an air ow rate of 1 l min1 .
The corresponding OTR during the respective microwell and
stirred tank fermentations can be calculated from Eq. (9):

5
N = 1000 rpm

4
3
2
1
0
0

(b)

2945

10

12

Time [hr]

Fig. 4. Microscale E. coli JM107:pQR706 fermentation kinetics at different

shaking speeds in 96-DSW plates: (a) LB medium; (b) LB-glycerol medium.
Error bars represent one standard deviation between OD measurements obtained from different wells.

shaped deep well is about twice that in a round well of the

same diameter. 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).
A value of kL a = 0.079 s1 can thus be estimated for microwell fermentations at N = 1000 rpm (kL a values calculated for
the lower shaking frequencies used in Fig. 4 are also shown in
Table 2(a)).
A relatively large amount of information has been published
on kL a measurements in mechanically stirred bioreactors. vant
Riet (1979) has reviewed a large number of investigations on
gasliquid mass transfer in stirred vessels and has correlated
the results to an accuracy of 2040%, depending on the specic
gassed power and the supercial gas velocity, vs . However,
estimation of the ungassed to gassed power ratio in a stirred tank
can be quite difcult due to the fact that previous investigations
have dealt with specic geometries and operating conditions.
Linek et al. (2004) obtained correlations for power input and
kL a in a vessel most similar to that used here (Tv = 0.29 m)

OTR = KL a(CL CL ),

(9)

where KL is the overall mass transfer coefcient, approximately

equal to the liquid-phase mass transfer coefcient, kL , in the
case of poorly soluble gases such as oxygen in aqueous systems; a is the specic gasliquid interfacial area; CL is the oxygen concentration in the liquid phase and CL is the saturation
concentration of oxygen. A typical value for oxygen solubility in aqueous systems at atmospheric pressure and T = 30 C
is around 1.2 mol m3 (Tromans, 2000). It is well known that
oxygen solubility may be signicantly decreased by the presence of ions and electrolytes in the fermentation media. The
aforementioned value, commonly used for water, was therefore corrected using the correlation provided by Quicker et al.
(1981). The oxygen partial pressure in the mixture was taken
into account and CL was estimated to be 0.19 mol m3 . The
maximum OTR (OTRmax ) will occur when DOT, and hence
CL , is close to zero. 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, respectively. 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.
In order to compare the results obtained in microwells and
in the stirred bioreactor under matched kL a conditions, fermentations in the 2 l bioreactor were subsequently carried out
at two agitation speeds, 700 and 1000 rpm, and at constant air
ow rate (1 l min1 ) using both LB and LB-glycerol media. At
N = 700 rpm, the calculated kL a of 0.077 s1 is similar to the
highest value obtained in microwells while at N = 1000 rpm
the kL a value of 0.17 s1 is signicantly higher (Table 2(a)).
Both biomass growth and DOT levels were measured during
the fermentations as shown in Fig. 5(a) and (b) for LB and
LB-glycerol media, respectively. With LB medium growth kinetics obtained at N = 700 and 1000 rpm were characterised by
similar values of  and maximum dry cell weights of 1.7 and
1.8 g l1 , respectively (Table 1(a)). However, the DOT measured at N = 700 rpm decreased after approximately 2 h, reaching a minimum value of 60%. In contrast, in the fermentation at
N =1000 rpm the DOT remained constant at 100% throughout.
Similar observations can be made for LB-glycerol fermentations (Fig. 5(b)). At the lower speed, the fact that DOT reached
approximately zero after 4 h of growth may suggest oxygen

M. Micheletti et al. / Chemical Engineering Science 61 (2006) 2939 2949

120

100

Biomass, N = 1000 rpm

Biomass, N = 700 rpm

DOT, N = 1000 rpm
DOT, N = 700 rpm

60

DOT [%]

40

0
4
Time [hr]

(a)

0
0

100

60

Biomass, N = 1000 rpm

Biomass, N = 700 rpm

40

DOT, N = 1000 rpm

DOT, N = 700 rpm

20

10

12

50

80

6
Time [hr]

(a)
120

Stirred bioreator (N = 700 rpm)

Shake flask (N = 300 rpm)

20

L-erythrulose concentration [mM]

Biomass [gDCW I-1]

Microwell (N = 1000 rpm)

80

Microwell (N = 1000 rpm)

Shake flask (N = 300 rpm)

40

Stirred bioreator (N = 700 rpm)

30

20

10

0
0
(b)

Biomass [gDCW I-1]

DOT [%]

Biomass [gDCW I-1]

2946

6
Time [hr]

10

12

Fig. 5. E. 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.

limitations begin at this point resulting in a slightly lower value

of  than that obtained in the corresponding microwell experiment. At the higher N = 1000 rpm the DOT values remained
constant at 100% throughout the process. At matched kL a values, the performance in both media was very similar at the
two scales. Identical  values (0.37 h1 ) were obtained in LB
medium in both geometries while similar max values were obtained in LB-glycerol medium. The maximum biomass concentrations obtained both bioreactors in LB (2.0 and 1.7 g l1 ) and
LB-glycerol (5.84 and 5.85 g l1 ) media were also very similar conrming kL a as a useful rst basis for scale translation
between the two geometries.
3.4. Scale translation of linked fermentationbioconversion
process
Finally, for the linked fermentationbioconversion process,
growth kinetics and L-erythrulose formation kinetics obtained
in microwell, shake ask and mechanically stirred bioreactors
are shown in Fig. 6(a) and (b), respectively, for LB-glycerol
medium. For the microwell (N = 1000 rpm) and mechanically

0
0
(b)

20

40

60
80
Time [min]

100

120

140

Fig. 6. Scale comparison of linked E. coli JM107:pQR706 fermentation

and bioconversion process kinetics at microwell (1 ml), shake ask (100 ml)
and stirred tank (1.4 l) bioreactor scales: (a) cell growth; (b) L-erythrulose
formation.

stirred (N = 700 rpm) bioreactors, fermentations were performed at matched kL a values as described previously. For the
shaken ask (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, the three biomass curves (Fig. 6(a))
exhibited similar values of max and Xnal as summarised in
Table 1(b). Likewise, 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 conrmed
by the specic activity values in Table 1(b). The corresponding
fermentations and bioconversion data, when the process was
repeated with LB medium, conrm the good agreement observed between microwell and mechanically stirred bioreactor
experiments under matched kL a conditions.
3.5. Scale-translation of hybridoma cultures based on power
consumption
The oxygen uptake rate of mammalian cells has been shown
to be in the range 2.101012 g cell1 h1 whereas for bacteria

M. Micheletti et al. / Chemical Engineering Science 61 (2006) 2939 2949

Viable cell count [cells mI-1]

1.4E+06
Shake flask (N = 120 rpm)

1.2E+06

Microwell (N = 120 rpm)

1.0E+06

Stirred bioreator (N = 150 rpm)

8.0E+05
6.0E+05
4.0E+05
2.0E+05
0.0E+00

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 0.46.2.33 103 g cell1 h1 .

The oxygen mass transfer coefcients required in mammalian
cell cultures are therefore signicantly lower, and generally
not considered limiting with regard to cell growth (Lavery and
Nienow, 1987). Consequently, for studies on antibody production in suspension mammalian cell cultures, a variety of bases
for scale-up have been proposed including uid turnover, vessel geometry, impeller tip speed and aeration rate (Chisti, 1993;
Varley and Birch, 1999; Xie et al., 2003). We, and others, have
considered power consumption as an initial basis for scale translation (Barrett et al., 2005; Hashimura et al., 2005) as this has
an impact on both mixing and mass transfer characteristics.
Table 2(b) shows parameters describing the ow characteristics of shaken microwells, shake asks and also a mechanically
stirred bioreactor used here for the culture of VPM8 hybridoma
cells. The liquid in both the shaken ask and microwell moves
in phase with the orbital motion of the shaker platform. This
was conrmed by visual observation. Considering power consumption, similar values ( 40 W m3 ) were found for shaken
ask and microwell while the prediction for the mechanically
stirred bioreactor is a factor of 10 lower at 3.64 W m3 (Barrett
et al., 2005). This analysis suggests than power consumption
can be used as an engineering basis to compare the results of
different shaken bioreactor geometries.
Fig. 7(a) shows the growth kinetics of VPM8 hybridoma
cells in microwell, shake ask and stirred-tank cultures. In all
three systems, the maximum cell densities were similar. After
approximately 3 days of culture, the viable cell density reached
1.0.1.1 106 cells ml1 . Maximum growth rates in the shake
ask and stirred tank were found to be almost identical at 0.046
and 0.048 h1 , respectively. However, the maximum growth
rate in the shaken microwell was slightly less at 0.040 h1 .
The yield coefcients for shake ask and mechanically stirred
bioreactor are similar (Barrett et al., 2005) and compare reasonably well with previously reported data (Ryu and Lee, 1997).
However, in microwells, a high value for Ylac/glu was found.
Although this could be the results of lactate production from
nutrients other than glucose (Sureshkumar and Muthanaransan,
1990), it suggests that cells are subjected to environmental
stresses in the microwell culture which might account for the
lower growth rate. Overall the similar growth proles obtained
for shake ask and microwell cultures suggest that matched
power consumption is, initially, at least, a good basis for translation between geometries and scales.
The corresponding antibody production kinetics are shown in
Fig. 7(b). In the case of shaken microwell cultures both the production rate and nal titre are signicantly higher than those in
the shaken asks and stirred bioreactor. The nal titre obtained
in microwells was 37 mg l1 , compared to 22 and 16 mg l1 in
the shaken ask and stirred tank, respectively. Similar results
have been reported by Girard et al. (2001). During a transient
transfection process, they found two-fold greater recombinant
protein expression by HEK 293 cells in agitated 12-well plates
compared to a 3 l stirred bioreactor. This increase in specic
antibody production at retarded growth rates, as found in microwells, is a common feature in industrial cell culture processes (Dinnis and James, 2005).

2947

25
20
15
10
5
0

20

40

60

Time [hr]

(b)

Fig. 7. Scale comparison of VPM8 hybridoma cells cultures at microwell

(0.8 ml), shake ask (100 ml) and stirred tank (3.5 l) bioreactor scales: (a) cell
growth kinetics; (b) antibody production. Error bars represent one standard
deviation between measurements obtained from different wells.

4. Concluding remarks
In this study, the potential for microscale process sequences
to quantitatively distinguish the performance of cultures grown
in different media was demonstrated. In addition, the fermentation and bioconversion kinetics of the whole cell biocatalyst
E. coli JM107:pQR706 were determined using different shaking frequencies. 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. By dening experimental conditions that
gave matched kL a values, it was shown that microscale results could be predictive of those obtained in shaken asks and
conventional bioreactor congurations at the laboratory scale.
Similarly, constant power consumption has been identied for
the initial scale comparison of mammalian cell cultures. Our
current work is applying these methods to the analysis of larger
libraries of evolved biocatalysts and mammalian cells.
Notation
a
ai
b

specic gasliquid interfacial area, m1 ; constant

in Eq. (6), mol l1
specic static gasliquid interfacial area, m1
constant in Eq. (6), s1

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. Micheletti et al. / Chemical Engineering Science 61 (2006) 2939 2949

bafe width, m
bond number (=
gd 2w /W ), dimensionless
constant in Eq. (4), m1
off-bottom clearance, m; constant in Eq. (1), dimensionless
oxygen concentration in the liquid phase, mol l1
saturation concentration of oxygen in the liquid
phase, mol l1
oxygen concentration uptake by the cells, mol l1
shaken ask diameter, m
shaken diameter, m
shaken microwell diameter, m
impeller diameter, m
oxygen diffusion coefcient, m2 s1
froude number (=ds (2N )2 /2g), dimensionless
gravitational acceleration, m s2
liquid height, m
liquid-lm mass transfer coefcient, m s1
overall mass transfer coefcient, m s1
impeller rotational speed, shaking frequency, s1
oxygen transfer rate, mol l1 s1
power consumption, W
gassed power consumption, W
modied power number, dimensionless
initial rate of product formation, mol l1 s1
Reynolds number (=
N d 2 /), dimensionless
Schmidt number (=/
DO2 ), dimensionless
time, s
bafe thickness, m
vessel internal diameter, m
temperature, C
supercial gas velocity, m s1
liquid volume, m3
wetting tension, N m1
constant in Eq. (4), dimensionless
biomass concentration, mol l1
constant in Eq. (4), dimensionless
constant in Eq. (6), mol l1

Greek letters
C

max




spacing between impellers, m

dynamic viscosity, kg m1 s1 ; specic growth
rate, s1
maximum growth rate, s1
liquid density, kg m3
liquid surface tension, N m1

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). Financial support from the 12 industrial partners supporting the BiCE
programme is also acknowledged. The authors would also like
to thank the UK Joint Infrastructure Fund (JIF), the Science
Research Investment Fund (SRIF) and the Gatsby Charitable

Foundation for funds to establish the UCL Centre for Micro

Biochemical Engineering. Support from the Biotechnology and
Biological Sciences Research Council is also acknowledged in
the form of a studentship for TB.

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