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Biotechnol. Appl. Biochem. (2006) 45, 1–12 (Printed in Great Britain)
REVIEW
Bioreactor systems for the production of biopharmaceuticals
from animal cells
James N. Warnock* and Mohamed Al-Rubeai†1
*Department of Agricultural and Biological Engineering, Mississippi State University, Mississippi State, MS 39762, U.S.A., and
†School of Chemical and Bioprocess Engineering, University College Dublin and Center for Synthesis and Chemical Biology,
Belfield, Dublin 4, Ireland
The demand for biopharmaceutical products is set to
see a significant increase over the next few years. As
a consequence, the processes used to produce these
products must be able to meet market requirements.
The present paper reviews the current technologies
available for animal cell culture and highlights the ad-
vantages and disadvantages of each method, while also
providing details of recent case studies. Processes are
described for both suspension and anchorage-depen-
dent cell lines.
Introduction
The key component for the commercial success of any
biopharmaceutical product is the ability to achieve large-
scale manufacture. Regardless of the efficacy of a drug, or the
efficiency of the downstream product purification, if there
is no process in place to produce the required amount of
the product to meet market demand, then it will fail. This
issue was recently highlighted by Anthony Fauci, Director
of the National Institute of Allergy and Infectious Diseases
(Bethesda, MD, U.S.A.), in his announcement that scientists
had successfully tested a human vaccine towards avian flu.
During his otherwise upbeat announcement, he remarked,
“The critical issue now is can we make enough vaccine,
given the well-known inability of the vaccine industry to
make enough vaccine?” The problem lies in the fact that
there has been an increase in the number and demand
for biopharmaceutical products produced from animal cell
cultures in recent years. This has led to an increase in the
number of contract manufacturing companies; however, this
is still insufficient to meet market demand, and the problem
will worsen as the number of approved biotherapeutics
increases. A part of the solution to this problem is to
increase awareness and understanding of the complex pro-
cess of producing biologics on a large scale. This will allow
those currently engaged in this field to exploit areas of
current weakness, while providing insight to those who are
not actively involved in process scale-up so that they may
doi:10.1042/BA20050233 1
be aware of the challenges faced with commercializing a
product.
Production capacity has significantly increased in the
last few years. Currently, many companies have a capacity
greater than 75 000 litres. In 2002–2003, only three compan-
ies had greater than 50 000 litres of mammalian cell culture
capacity. According to an industry survey, the produc-
tion capacity for biopharmaceutical manufacturing will ex-
pand by an average of 48 % over the next 5 years for mam-
malian and microbial production systems. Approximately 35
biopharmaceutical products are currently on the market,
and an estimated 700 biotherapeutics are in clinical develop-
ment, with 150–200 in late-stage trials [1]. Obviously, the
requirements in terms of process development and bio-
reactor capacity are tremendous. There is a total of approx.
92 companies operating Good-Manufacturing-Practice faci-
lities manufacturing biopharmaceutical products using mam-
malian cell culture for clinical trials and in-market supply
located in U.S.A. and Europe. The worldwide mammalian
cell culture capacity for Phase III and in-market supply of
biopharmaceuticals was estimated to be 1.7 million litres at
the beginning of 2004, rising significantly in 2008 [2].
The present review will discuss the traditional and
newly developed bioreactor technologies available to pro-
duce biologics using animal cell culture on a large scale. Tra-
ditional systems are tailored towards anchorage-dependent
cell lines, but in most cases can be used for suspension cells.
As more and more cell lines are adapted to serum-free
suspension culture, recent advancements have focused on
cell retention in bioreactors to allow for perfusion methods
Key words: animal cell biotechnology, biopharmaceuticals, bioreactor system,
filtration system, microcarrier, perfusion culture.
Abbreviations used: ATF, alternating tangential flow; BHK cell, baby-hamster
kidney cell; CHO cell, Chinese-hamster ovary cell; ECS, extracapillary
space; EPO, erythropoietin; HFBR, hollow-fibre bioreactor; ICS,
intracapillary space; IFN-γ, interferon-γ; IL-2, interleukin-2; IP, infectious
particles; mAb, monoclonal antibody; OUR, oxygen uptake rate;
STR, stirred tank reactor.
1
To whom correspondence should be addressed (email
m.al-rubeai@ucd.ie).


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2 J. N. Warnock and M. Al-Rubeai

Table 1 Characteristics of solid microcarriers divided into solid and macroporous carriers. Solid micro-
Particle
carriers have a lower surface area than macroporous car-
Microcarrier Manufacturer Material diameter (µm) riers, and can be susceptible to bridging when cells reach
high densities. Bridging occurs when cells attached to one
Cytodex 1 Amersham DEAE-dextran 147–248
Biosciences
carrier come into contact with another carrier and form a
Cytodex 2 Amersham THMAP–dextran 135–200 bond between the two. As a consequence, carrier aggre-
Biosciences gates form, which inhibits the efficiency of the reaction.
Cytodex 3 Amersham Gelatin–dextran 141–211
Biosciences
Macroporous carriers have a higher surface area and
FACT III SoloHill Cross-linked polystyrene 90–125 therefore have a greater potential for cell growth. It has been
Hillex (H) SoloHill Modified polystyrene 90–212 shown that Vero cells reach densities of 1.05 × 106 cells/ml
Biosilon Nunc Polystyrene 160–300
2D MicroHex Nunc Polystyrene 125 × 25
and CHO cells (Chinese-hamster ovary cells) can reach
densities of 2.16 × 106 cells/ml [6]. Similarly, V79 cells that
were genetically designed to express human cytochrome
P450 reached a density of 107 cells/ml after 11 days, resulting
to be used. Finally, several manufacturers have developed in a total of 3.6 × 1010 cells [7]. Unfortunately, cell densities
disposable bioreactors to simplify regulatory processes. are often limited by mass transport into the particles. As
the cell numbers increase, nutrients are unable to diffuse
into the centre of the carrier, while toxic metabolites, such
Anchorage-dependent versus as CO2 and lactate, are unable to diffuse out into the bulk
suspension cells medium. This leads to the evolution of a necrotic zone within
the particle. In addition, cell products are trapped within the
At the laboratory scale, or for high-throughput screening particle and cannot be harvested, which also leads to a
or monitoring purposes, it is often more desirable to use loss in product yield [8]. As a result, product titres from
anchorage-dependent cells than suspension cells. However, solid microcarrier cultures are often higher than those for
this poses problems with scale-up, as the amount of available macroporous cultures, as illustrated by Berry et al. [9]. The
surface area becomes limited. Several strategies have been characteristics of commonly used microcarriers are pre-
developed to maximize the amount of available surface area. sented in Tables 1 and 2.
To produce the required amounts of product for clinical In order to overcome the problems associated with
trials, scale-up is often performed in roller bottles. However, large-scale cultivation of anchorage-dependent cells, many
roller bottles have a finite surface area and scale-up can only cell lines have been successfully adapted to grow in suspen-
be achieved by increasing the unit number of bottles, which is sion culture. Scale-up of these systems is relatively simple,
extremely labour-intensive and susceptible to contamination and the maximum cell densities achieved in fed-batch
[3]. Modern facilities have overcome these problems by the cultures can be as high as 2 × 107 cells/ml. Suspension cells
use of expensive robots; however, it is still not possible to can be grown in either STRs (stirred tank reactors), similar
control environmental conditions such as pH or dissolved to those used in microbial fermentations, which are relatively
oxygen levels [4]. The most successful method for the scale- easy to scale-up beyond 10 000 litres, or airlift reactors,
up of anchorage-dependent cells has been the use of micro- which are more commonly used in wastewater treatment.
carriers, first reported by van Wezel [5], which provide a The simple design and operation, along with flexibility and
high surface-area-to-volume ratio and can be used in trad- ease of licensing, make these systems popular for manu-
itional stirred-tank bioreactors. Microcarriers can be sub- facturing.

Table 2 Characteristics of macroporous microcarriers

NA, data not available.

Microcarrier Manufacturer Matrix Diameter (mm) Pore size (µm) Porosity (%)

Cultisphere-G, S, GL Percell Biolytica Gelatin 0.17–0.5 ∼ 50 50

Cytoline 1, 2 Amersham Biosciences Polyethylene and silica (1.7–2.5) × (0.4–1.1) 10–400 65
Cytopore 1, 2 Amersham Biosciences Cellulose 0.2–0.28 30 95
Fibra-cel New Brunswick Scientific Polyester non-woven fibre and polypropylene 6 NA NA
ImmobaSil FS, D, HD Ashby Scientific Silicone (HD + 316L stainless steel) 0.8 × 0.3 (FS, HD) 50 × 150 > 40
10 × 1 (D)
Microsphere Cellex Collagen 0.5–0.6 20–40 75
Siran Schott Glasswerke Glass 0.3–5 10–400 60

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Figure 1 Factors that cause cell death in large-scale animal cell culture [97]
Stirred tank bioreactors
Although many alternatives have been developed, the tradi-
tional STR has emerged as the industry’s technology of
choice [10]. The attractiveness of STRs stems from their
simplicity, and the ease of monitoring and controlling scale-
up. They are able to provide efficient gas transfer to cells
and they have a long history of use with a variety of micro-
organisms. Mixing in an STR is achieved by means of an impel-
ler, which is typically axial flow as opposed to a Rushton tur-
bine, the common choice for microbial fermentations. The
impeller speed will be sufficiently high enough to ensure that
each phase of the vessel contents is of uniform composition,
i.e. they do not vary with position inside the reactor. Con-
sequently, STRs are homogenous systems when used at
smaller scales. When the system is scaled up, difficulties in
homogeneity within the reactor can occur. The main mech-
anism of cell death in bioreactor culture is apoptosis. It can
be induced by exhaustion of nutrients, an absence or an
excess of dissolved oxygen, gross excursions from the ideal
physiological environment (e.g. extreme pH) and physical
abuse (see Figure 1). The mixing regimen has to be optimized
in large-scale systems to alleviate these environmental pro-
blems and ensure the dissolved oxygen concentration is the
same throughout the liquid bulk phase [11]. This prevents
the development of localized areas of either high or low
dissolved oxygen concentrations, which are detrimental to
cells, and the formation of dead zones. The disadvantage of
such rigorous agitation is that high mechanical forces, such as
shear stresses, exist within the vessel. The eddies generated
by mixing do not cause direct damage to animal cells in
suspension, provided that their microscale of turbulence is
greater than the cell diameter. Direct gas sparging is generally
the most damaging thing to cells, the extent of the damage
decreasing with reduced sparging rate until it reaches below
0.2 vvm (volume · volume−1 · min−1 ), where it becomes equi-
valent to unsparged conditions [12]. Cell damage can occur
Bioreactors for cell culture 3
Figure 2 Scanning electron micrograph of CHO cells immobilized on
ImmobaSil FS macroporous microcarriers (scale bar, 50 µm)
through stresses created by bubble entrainment and bubble
bursting [13]. Highly agitated cultures will form vortices
which will increase the rate of bubble burst; thus the
hydrodynamic forces that result from increased agitation
have an indirect effect on cell viability [14].
Many cell lines that are cultivated in STRs have been ad-
apted to serum-free suspension culture. Regulatory author-
ities have encouraged the development of processes that do
not include materials of animal origin and, therefore, many
companies have developed defined serum- and protein-
free media. In the absence of serum, and therefore fibro-
nectin, cells do not attach to a solid substrate and can be
adapted to grow in suspension. Suspension cultures can
be easily scaled-up as the surface area to volume ratio is
no longer a factor. Although STRs are commonly used for
suspension cell culture, the same bioreactors can be used
for anchorage-dependent cell lines that do not survive
in suspension. These cells can be cultivated on solid or
macroporous microcarriers. It is also possible to entrap sus-
pension cells within macroporous beads to increase the cell
density. Macroporous microcarriers offer protection from
hydrodynamic forces, provided that cells are able to migrate
towards the centre of the particle. Many fibroblasts can
move as quickly as 1 cm a day on a smooth surface [4], but
the internal surface of these matrices is rarely flat or smooth
and is quite tortuous for cells. However, once cells are pro-
tected from external forces, the mixing of the stirred tank
can be increased. The resulting high cell densities range from
1 to 6 × 107 cells/ml [15–17], depending on cell line and
microcarrier used. Figure 2 shows CHO cells that have been
grown to a high density on ImmobaSil FS microcarriers.
Stirred tanks have been successfully used in the produc-
tion of several biotherapeutic products, including mem-
brane-anchored recombinant proteins, urokinase-type
plasminogen activator, prourokinase, IgG and IgA mAbs


C 2006 Portland Press Ltd
4 J. N. Warnock and M. Al-Rubeai
Figure 3 Schematic representation of an airlift bioreactor with internal
recirculation (left panel) and external recirculation (right panel)
(monoclonal antibodies) and vesicular stomatitis virus
[15,18–22].
The largest stirred tank bioreactors available are
20 000 litres, which are operated by Lonza Biologics in
Portsmouth, NH, U.S.A. To our knowledge, fed-batch mode
is the most common approach in industry for the production
of biopharmaceuticals from mammalian cells.
Airlift bioreactors
Although STRs are considered to be the industrial standard
for animal cell biotechnology, airlift bioreactors have been
used in a number of large-scale processes. Most notably,
airlift reactors are used by Lonza Biologics, the world’s
largest contract manufacturer of mAbs. Airlift bioreactors
can take on two configurations, namely internal-loop or
external-loop, as shown in Figure 3. These reactors offer
some advantages over STRs, such as being more energy-
efficient, not having a mixer shaft and having a less harsh
hydrodynamic environment. The lack of a mixer shaft eli-
minates one source of potential contamination, albeit one
of low risk. However, the lack of an impeller is a distinct
advantage. The rigorous agitation required to provide
a homogeneous environment in an STR presents three
possible mechanisms of cell damage. The first is direct
Table 3 Comparison of batch, fed-batch and perfusion culture systems
Stirred batch (batch/fed-batch)
Culture type Suspension
Cell concentration Low/medium
Productivity Low/medium
Specific cell productivity Low/medium
Scale-up Easy
Operation Simple
Monitoring and control Direct and easy
Operating cost Low


C 2006 Portland Press Ltd
contact between cells and the impeller blades, the second
is contact between cells and other stationary bioreactor
components, such as the baffles, and the third arises from
bubble burst caused by the vortices created by the impeller,
as mentioned in the previous section. In an airlift bioreactor,
these problems are eliminated by the absence of an impeller.
Furthermore, the recirculation of the cells and medium
reduces the exposure of cells to surface bubble burst.
As with STRs, airlift bioreactors can be used to cultivate
either suspension cells [23] or anchorage-dependent cells
cultivated on microcarriers [24]. It has been recently shown
that BHK-21 cell (baby-hamster kidney 21 cell) cultivation
in protein-free medium is feasible in 1000-litre airlift bio-
reactors. The cells had a higher metabolic rate than in a
bubble-free aerated STR; however, this may have been due
to an increase in cell stress, as specific IL-2 (interleukin-
2) productivity was significantly lower in the airlift reactor
cultures, relative to the STR, and lactate dehydrogenase was
higher in airlift bioreactors when compared with the STR
[25]. Airlift bioreactors have been scaled-up to 2000 litres
and have been routinely used in a fed-batch mode to
produce mAbs for therapeutic and diagnostic use. Further
information about the recent advances in airlift bioreactors
can be found in the review by Merchuk [26].
Filtration systems
STRs can be run in three different modes, namely batch,
fed-batch or perfusion, and each has its own advantages and
disadvantages [27]. Fed-batch systems are widely used in in-
dustry as they can provide greater cell densities and product-
ivity compared with batch reactions. The advantage of per-
fusion cultures is that cells are retained in the reactor
while the product is continuously harvested; therefore high
productivities can be achieved in a smaller bioreactor than
for batch or fed-batch systems [28]. However, for perfusion
reactions to be efficient, and a viable option for industrial
production, a good separation method is required to retain
cells in the vessel while simultaneously harvesting the pro-
duct. A comparison between batch and perfusion cultures
is presented in Table 3. Separation of cells and product on a
Hollow fibre Spin-filter and dialysis membrane
Stationary Suspension
High Medium
High High
Insufficient data High
Impossible Easy
Complex Relatively simple
Indirect and complex Direct and easy
High Low

large scale is based on the physical properties of the parti-
cles, and the different methods utilize either size or
density.
Spin filters
Of all the cell retention methods currently used, the spin
filter is probably the simplest and the oldest device. The
spin filter retains cells by use of a wire mesh that rotates
in the centre of the reactor vessel. The steric effect of the
rotating screen allows cells to be retained when the screens
have pore sizes larger than the average cell [28]. Spin filters
typically are made of 316L stainless steel and have pore sizes
that range from 10 to 25 µm [29–31], although larger pore
sizes of 50 µm and even 120 µm have been successfully
used to retain cell aggregates [32] and microcarrier cultures
[33]. In addition to allowing cell separation from product in
perfusion cultures, the use of a spin filter has been proven
to reduce hydrodynamic damage to cells; consequently, cell
viability and productivity are improved when compared with
cultures performed in the absence of a spin filter [34]. The
primary concern when using a spin filter is fouling or clogging
of the screen. There are two different mechanisms behind
clogging [29]; the first is when the filtration flux exceeds the
retention capacity of the filter screen and cells accumulate in
the pores. This can occur suddenly. The second mechanism
occurs at a much lower rate and is only apparent in long-
term cultures that exceed 40 days. Cells attach to the screen
and begin to grow. Once they reach a certain density, they
will block the pores, preventing the product from being
removed from the vessel.
Spin filters have been successfully used in numerous
studies that have investigated biotherapeutic production
from animal cells. The performance of a spin filter can be
enhanced by optimizing the culture conditions and perfusion
parameters. Spin filter rotation speed and perfusion rate do
not affect the retention of viable cells; however, enlarging cell
diameters and the formation of aggregates during opera-
tion of a perfusion culture increase cell-retention rates
from 75 to 95 % [30]. Additionally, the cell retention per-
formance of a spin filter has been shown to be comparable
with that of an ultrasonic filter. When a hybridoma bcl-2 cell
line was cultivated in both systems, the maximum cell num-
bers achieved were 1.21 × 107 and 1.58 × 107 cells/ml for the
ultrasonic filter and the spin filter respectively [35]. Corres-
pondingly, when hybridoma cells were cultivated using a
medium supplemented with 1 % newborn-calf serum and
peptone, the maximum cell number maintained in steady
state was 1.3 × 107 cells/ml [36]. Another consideration is
scale; the largest ‘off-the-shelf’ spin filter is suitable for a
50-litre bioreactor. Consequently, the use is limited to pro-
duct research and development studies and they are not
used for commercial manufacture.
Bioreactors for cell culture 5
Dialysis perfusion
The use of a dialysis membrane is a simple yet effective way
of performing a perfusion culture. The membrane allows
for the retention of high-molecular-mass components, such
as antibodies, as well as cells, while low-molecular-mass
components and metabolic waste products can be conti-
nuously removed. Dialysis operates on the principle of
mechanical or osmotic pressure differences being present,
allowing for the net transport of molecules up to a certain
size to cross the membrane. The molecular cut-off of the
membrane allows for the selectivity of the system. The sys-
tem has several advantages over other perfusion methods;
the product can be retained in the bioreactor and is sub-
sequently recovered at a far greater density, which simplifies
the purification process. Dialysis tubing can be easily re-
trofitted to standard STRs, and expensive consumables,
special equipment or technically challenging operations are
not required. Finally, system homogeneity allows for simple
reactor control and assessment of the cellular physiology
[37].
Several groups have evaluated the dialysis perfusion
system as either an internal or external culture system.
The internal method cultures hybridoma cells inside the
dialysis tubing, which is suspended in medium [38,39]. Un-
fortunately, the cell compartment is not well mixed and
consequently the maximum usable volume is limited. The
external method effectively turns the system inside out
with the cells being cultured in the reactor vessel and the
perfusate flowing through the dialysis tubing. This provides
a large, well-mixed cell compartment and a small medium
compartment that is attached to a larger medium reservoir
[37]. These systems have been scaled up to 1000 litres [40].
The length (and therefore the surface area) of the tubing
has been shown to be an important parameter that affects
the maximum viable cell number. Despite the potential
application of dialysis perfusion systems, they have not been
utilized in industry. Very few recent developments have
been made and they are considered an old-fashioned techno-
logy.
HFBRs (hollow-fibre bioreactors)
HFBRs operate on the same principle as dialysis perfusion
systems. In this bioreactor, cells attach to the outer surface
of semi-permeable fibres, growing in the ECS (extracapil-
lary space) while medium is circulated through the ICS
(intracapillary space) or lumen. Nutrients diffuse through
the fibres, usually made of cellulose acetate, while toxic
metabolites diffuse into the ECS and are carried away from
the cells. A unit consists of thousands of fibres traditionally
housed in a cylinder, but recent devices house fibres in a
cartridge. The product accumulates within the ECS and can
be harvested intermittently.


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6 J. N. Warnock and M. Al-Rubeai
There are numerous advantages to using HFBRs and
they have received attention for the commercial production
of mAbs and recombinant proteins [41–43]. The main ad-
vantage of HFBRs is the high cell densities that can be
achieved. Numerous studies have reported near tissue
densities of > 108 cells/ml [41,44]. These high densities in
turn lead to increased production and concentration of
products and enhance the specific productivity in a relatively
small bioreactor [45–47]. However, HFBR systems are
associated with large diffusional gradients that limit scale-
up and affect product quality. As medium enters the lumen,
it encounters a positive transmembrane pressure and is able
to permeate into the ECS. As this pressure decreases along
the length of the fibre, it becomes negative towards the
outlet, causing medium to flow back into the ICS. Despite
the superior cell densities, HFBR systems are not used in
commercial processes. However, they have shown potential
in the development of a bioartificial liver assist device [48].
Acoustic filters
It has been suggested that acoustic retention devices have
the greatest potential of all the cell separation systems [49].
Unlike several other separation devices, acoustic filters have
no physical barrier and no mechanical parts and are there-
fore less susceptible to fouling or mechanical failure [50].
Acoustic cell separators generate a fixed ultrasonic wave
field that causes cells to form reversible aggregates at the
pressure node of the field [51,52]. Differences in size, density
and compressibility allow the separation of cells from the
product or medium [53] or the retention of viable versus
non-viable cells [51,54]. On the small scale, the acoustic
filter has been shown to be a reliable and simple cell reten-
tion device for long-term cultures [55]. Furthermore, the
system has been successfully scaled-up to 200 litres/day
perfusion cultures, showing comparable performance with
small-scale systems. On a large scale, the performance of
the separator was optimized and provided 96 % separation
efficiency [54].
The main concerns associated with acoustic cell reten-
tion devices are the increased shear stress that cells are
exposed to during pumping and the pressure differences
present in the separator [50,56]. These can lead to a de-
crease in cell viability, cell-specific productivity or product
quality, especially in shear-sensitive insect cells [57]. To over-
come the problem of shear stress, it has been proposed
that cells be back-flushed, allowing shear-sensitive cells to
be perfused with the separator. The original method of
back-flushing used clarified medium [57]. However, once
the medium has been used, it needs to be clarified again,
causing an unfavourable increase in the harvest pump
flow to maintain a constant average perfusion rate [56].
The alternative method is to use air back-flush. Results


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presented by Gorenflo et al. [56] were able to demonstrate
that an air-back-flush system was able to eliminate recircu-
lation pumping of cells, thus eliminating the potential of shear
stress damage, while maintaining separation efficiencies
above 90 % at perfusion rates of 10 litres/day and 107 cells/
ml. An additional drawback of acoustic filtration systems
is that they are an external device and, when cells are in
the external loop, they are exposed to uncontrolled con-
ditions and nutrient concentrations for short periods of
time [58]. It is not known how the cells respond to these
relatively short periods in the external loop and it is not
thought to be of great detriment. Nonetheless, air back-
flush eliminates the need to pump cells and allows the selec-
tion of a separator residence time that is suited to the sens-
itivity of the particular cell line [56]. Most of the studies on
acoustic filters have been conducted on a small/laboratory
scale. Success at the industrial scale would be dependent on
their ability to achieve high separation rates under high flow
conditions [49]. Filters can have up to 95 % efficiency with
perfusion rates of 200 litres/day. Beyond this, separation
efficiency shows a sharp decline. To overcome this, larger
retention devices are needed, which in turn require a higher
power input [54]. Further details of acoustic filter retention
technology can be found in the recent review by Shirgaonkar
et al. [49].
Cross-flow filtration
Cross-flow filtration has been widely tested in industry, and
may offer a good alternative to spin filters or acoustic devices
on a larger scale. The principle behind cross-flow filtration
is to force the cell suspension to flow tangentially to a mem-
brane with the product crossing the membrane to allow
for harvest. Cross-flow filtration was originally developed
to reduce the fouling of membranes by particle deposition
in pores and concentration gradients of macromolecules,
which causes aggregation and gel formation [28]. Ironically,
despite membrane rinsing or back-flushing, fouling and
clogging problems have been frequently reported [59–61].
However, by incorporating an ATF (alternating tangential
flow) device, fouling is avoided by washing cell aggregates
back into the bioreactor. The system works by alternatively
pulling culture medium into the ATF inner chamber via the
movement of a diaphragm at the top of the unit and pushing it
back into the reactor. The back flow part of the cycle causes
a small back-flush to occur across the filter membrane,
cleaning the filter pores and surface. Furthermore, the
ATF uses a low-shear, large-surface-area diaphragm, which
reduces cell stress. This system was successfully used for
the production of recombinant proteins in a microcarrier-
based culture. By using such a device, Bleckwenn et al. [62]
reported that the exponential growth phase of the cells
was increased by 50 h when compared with batch culture.

This had the effect of increasing the maximum cell density
from 1.5 × 106 to 4.4 × 106 cells/ml [62]. The ATF system
has been used at the 1000-litre scale.
Fixed and fluidized-bed systems
STRs and airlift bioreactors are well suited to suspension cell
cultures. However, not all industrial cell lines can be adapted
to suspension culture, and these cells are strictly adherent.
Thus, when using anchorage-dependent cells to produce
biologics, it may be more appealing to use high-density/low-
volume systems as opposed to high-volume/low-density
systems such as microcarrier STRs. The alternative methods
for the production of biologics are the fixed and fluidized-
bed systems.
Packed-bed bioreactors
Packed-bed bioreactors have been used for the cultivation
and production of a wide range of cell lines and biologics.
Some recent examples include mAbs, anti-leukaemia factor
from stromal cells, recombinant proteins (e.g. recombinant
Ca2+ -binding receptor and HIV-1 gp120), and retrovirus
vectors for use in gene therapy [15,63–67]. A disadvantage of
microcarrier culture in stirred tanks is that cells detach from
carriers at high densities and, along with cell debris, cause
complications in product purification during downstream
processing. Packed-bed bioreactors have the advantage that
they are capable of generating high cell densities with a low
concentration of free cells in suspension. This is possible
due to the low shear forces present in the system. Cells
are immobilized within porous carriers that may be porous
ceramic beads [68], porous glass beads [69] or polyester
discs [15,64,67], which are packed and retained in a
cylindrical vessel through which culture medium is recircu-
lated. A characteristic problem in intensive production
systems is the reduced transport rate of limiting nutrients,
such as oxygen to immobilized cells, which restricts their
final density. Packed-bed technology has overcome this
problem by the use of intraparticle convective flow. Through
the development of a simple hydrodynamic model based
on the Blake–Kozeny equation, Park and Stephanopoulos
[70] significantly improved the transport of oxygen in a
packed bed, allowing the maintenance of cell viability and
productivity while sustaining a low-shear environment. In
their evaluation of the model, they achieved cell densities of
5.1 × 108 cells/ml. They also calculated the specific insulin
productivity to be 0.88 × 10−5 microunit · cell−1 · h−1 . The
model developed allows packed-bed bioreactors to be
scaled-up severalfold before oxygen becomes limiting. Any
oxygen limitations that do occur primarily exist close to
the bioreactor outlet. Another development to overcome
this problem has been to provide oxygenation to the bed
Bioreactors for cell culture 7
Figure 4 Schematic representation of the CelliGen Plus packed-bed
bioreactor (courtesy of New Brunswick Scientific; www.nbsc.com)
through silicone tubing. This simple but effective method has
been used for the successful culture of hepatocytes and the
production of retrovirus particles from the human packaging
cell lines FLYRD18 and TEFLYRD [71,72].
An alternative system is the CelliGen® packed-bed
bioreactor, developed by New Brunswick Scientific (Edison,
NJ, U.S.A.), which is shown in Figure 4. Conventional
packed beds require a conditioning vessel to control culture
parameters such as temperature, dissolved oxygen and pH.
The culture medium is circulated through the packed bed
and the product may be concentrated and continuously
or periodically harvested from the conditioning vessel for
prolonged periods. An important parameter in the optimiz-
ation of these arrangements is the circulation rate; if the rate
is too low, then gradients in pH, nutrients and metabolites
will appear along the bed, affecting cell viability, productivity
and product quality, as can be seen in HFBRs [73]. If the
circulation rate is too high, it may have an adverse effect
on cell attachment or product formation. This problem is
eliminated in the CelliGen® packed-bed bioreactor by the
fact that the bed is contained in a basket within a stirred tank
bioreactor. Mixing in the bioreactor is achieved with either
a cell-lift impeller, which allows bubble-free medium to flow
through the packed bed [67] or a double screen concentric
cylindrical cage impeller. Shi et al. [74] have shown that the
latter impeller is able to increase convective mass transfer,
cell concentration and mAb product concentration. The
significant improvements were attributed to the increased
surface area, allowing for convective oxygen transfer and
protection of cells from hydrodynamic stresses. Both
methods create a homogeneous environment for cells to
grow and product can be directly removed from the culture
vessel, which allows for easier downstream processing and
product purification.


C 2006 Portland Press Ltd
8 J. N. Warnock and M. Al-Rubeai

A disadvantage of packed-bed bioreactors is the inac-

cessibility of carriers, making it difficult to monitor viable
cell number. Cellular metabolism can be followed by online
measurements of pH and dissolved oxygen and offline deter-
minations of glucose, lactate, ammonia and glutamine. The
substrate uptake rates and production rates are con-
ventionally used as cell growth markers [75]. In general,
the cell concentration has a linear correlation with glucose
uptake rate at high cell densities. Thus it is possible to calcu-
late the latter from the former [76]. However, this yield
coefficient is not a true constant and will vary for each cell
line and for each bioreactor system. Therefore it should
be determined before being used to estimate cell number.
Alternatively, cell number can be determined through
measuring the OUR (oxygen uptake rate). By placing oxygen
probes upstream and downstream of the packed bed and
measuring the percentages of dissolved oxygen, it is possible
to determine the OUR, which can be directly correlated
with cell number. The density of cells within an immobilized
culture is an important parameter for many purposes, includ-
ing the calculation of the specific productivity of a system.
Figure 5 Schematic representation of the Cytopilot mini fluidized-bed
bioreactor
Fluidized-bed bioreactors
Abbreviation: dO2 , dissolved oxygen; Temp, temperature.
The most widely used culture system for porous micro-
carriers is the fluidized-bed bioreactor [77]. The principle
of their operation is that microcarriers of higher density
than the culture medium are suspended by the upward flow stirred culture. In contrast, the production rate of hy-
of the medium, which is circulated through the bed. The bridoma cells cultured in the fluidized bed remained appre-
height of the bed will increase as fluid flow increases. Medium ciably higher than that in stirred-tank culture. The difference
circulation and fluid flow through the bed are achieved by was assumed to be caused by BHK cells changing their
using either a peristaltic pump or agitation. The advantage of production characteristics during long-term cultivation. This
this system is that carriers are separated from direct aer- phenomenon had previously been reported in hybridoma
ation and agitation, which avoids local high shear forces. In cells [80]. An alternative reason is that, unlike hybrid-
addition, fluidized-bed bioreactors provide a very high mass- oma cells, specific cell productivity is related to specific
transfer interface, since both cells and fluid are moved [78], growth rate. It has been shown by Leelavatcharamas et al.
making it a suitable production system on the larger scale. [81] that there is a linear relationship between the pro-
Commercially available systems are available up to 400 litres, duction of IFN-γ (interferon-γ ) and the specific growth
which can produce cell densities of 2 × 108 cells/ml. There rate of CHO cells. Therefore, in long-term cultures where
are not currently any licensed production processes that use the cells remain in the stationary phase over an extended
fluidized-bed bioreactors, as is also the case with packed-bed period, productivity will be low as the specific growth rate
bioreactors. is essentially zero.
Earlier studies into the potential of fluidized-bed react- Another substrate for the immobilization of cells
ors for the production of biologics focused on the use grown in fluidized-bed bioreactors is polyethylene and silica
of porous glass beads. These studies conducted by Kratje microcarriers such as Cytoline (Amersham Biosciences,
et al. [77,79] examined the potential of fluidized-bed react- Uppsala, Sweden). These have been successfully used in the
ors as production systems using hybridomas suspension cells production of recombinant human proteins, especially hu-
and anchorage-dependent BHK cells for the production of man IFN-γ and EPO (erythropoietin) [82,83]. These micro-
IL-2 (interleukin-2). In these studies, cell densities of 3.8 × carriers have been specifically designed for use in the Cyto-
108 cells/ml intrasphere volume were reached, while the IL-2 pilot fluidized-bed bioreactor (Figure 5) and have proved to
production rate was 0.75 mg · l−1 · day−1 . In spite of the cell offer great potential for the production of biologics. Kong
density being over 18-fold higher, the productivity showed et al. [84] compared CHO cell cultures in this system with
a 1.9-fold decrease when compared with a homogeneous solid microcarrier culture in a stirred-tank bioreactor and


C 2006 Portland Press Ltd

reported that the product output rate was approximately
three times higher in the fluidized bed, with the specific
production rate being 5.5 times higher. The volumetric pro-
ductivity of the stirred tank reached 5.6 mg · l−1 · day−1 during
the growth period. However, after the maximum cell
density was reached, the volumetric productivity decreased
and was maintained around 1.65 mg · l−1 · day−1 . In contrast,
the fluidized-bed bioreactor maintained a consistent volu-
metric productivity of 22.8 mg · l−1 · day−1 . It was suggested
that the isolated aeration and agitation in the fluidized-
bed bioreactor reduced the amount of hydrodynamic and
mechanical stress imposed on the cells. Thus more cellular
energy was utilized in product formation, resulting in the
higher and more consistent productivity. The total product
yield was reported as 34.2 and 13.2 mg/day for the 2-litre
fluidized-bed bioreactor and the 10 litre stirred tank bio-
reactor respectively. The Cytopilot has also demonstrated
good retroviral vector production, with a maximum virus
titre of 107 IP (infectious particles)/ml, and a total viral yield
of 5.03 × 1010 IP in 11.4 litres. In roller-bottle cultures, daily
titres of 5 × 106 IP/ml were achieved from 50 ml per bottle
(total yield: 2.5 × 108 IP per bottle). Therefore the total yield
obtained from the Cytopilot over 19 days was equivalent to
the yield from ten daily harvests from 20 roller bottles [85].
It was observed that virus titre increased with an increasing
cell number. Therefore, as the Cytopilot has the potential for
higher cell numbers in longer runs, it also has the potential
for greater virus titres.
When comparing the number of cells in each square
metre of microcarrier surface area, the fluidized-bed bio-
reactor had a value five times lower than that of the STR.
This shows that there is further potential to increase the cell
number and improve the productivity of the system. In order
to unlock this potential, it is necessary to ascertain the limit-
ing factor for growth and how to overcome this limitation.
Investigations carried out by Preissmann et al. [78] deter-
mined that the transport of oxygen to cells within the
macroporous matrix cultivated in a fluidized-bed bioreactor
was severely limited, leading to poor process performance.
Although no limitations in nutrient concentration were
detected, the beads were not believed to be an ideal sub-
strate to grow cells to tissue-like densities.
Efficient glycosylation is an important factor in the
development of a bioprocess production method. The exi-
stence of gradients that cause decreases in dissolved oxygen,
nutrients or pH may have implications for product quality,
as well as cell densities. In a recent study, the relative pro-
portion of glycosylation site occupancy for IFN-γ was not
seen to change over time during a 500 h culture. Hence, it
was concluded that long-term perfusion cultures of CHO
cells in a fluidized-bed bioreactor could produce IFN-γ with
a consistent and highly comparable degree of glycosylation
[82]. Further studies, carried out by Wang et al. [83], have
Bioreactors for cell culture 9
also examined the productivity and quality of EPO from a
recombinant CHO cell line, cultured in a Cytopilot fluidized-
bed bioreactor. EPO is particularly vulnerable to a decline
in sialylation due to the extensive level of glycosylation and
heterogeneity. It was therefore important to show that the
glycosylation profile of EPO in the Cytopilot did not differ
from other culture systems. Results have shown that no
difference was observed in electrophoretic product profiles
between fluidized bed, stirred tank and stationary cultures.
In addition, the significantly higher productivity rate in the
fluidized-bed bioreactor, when compared with STRs, makes
it an attractive option for the large-scale production of re-
combinant proteins.
Disposable bioreactors
Disposable bioreactors, such as the CellCube (Corning),
the Cell Factory (Nunclon) and the Wave bioreactor (Wave
Biotech), are gaining increasing popularity for industrial
applications. This is because they offer many benefits, which
include low initial investment costs, reduced lifetime oper-
ating costs, reduced validation, simplified scale-up and low
risk of cross-contamination. The CellCube culture system
has been successfully used for the production of retrovirus
vectors, mAbs and recombinant proteins [66,86,87]. The
performance of the CellCube is reported to be comparable
with the CelliGen® packed-bed reactor, with each system
producing an average of 2.1 × 107 and 0.55 × 107 IP/ml res-
pectively [88]. The yield from the CellCube was also
reported to be comparable with that from the automated
roller-bottle system, RollerCell 40 [87]. When the CellCube
was run under Good-Manufacturing-Practice conditions, the
virus titre was between 1.2 × 106 and 3.0 × 106 IP/ml. This
was lower than titres recorded under test conditions, owing
to a higher perfusion rate. While the higher perfusion rate
reduced the vector concentration, it did increase the titre
stability and thus the quality of the vector preparation.
The disadvantage of the CellCube bioreactor is that some
components, such as the oxygenator and the probes, must
be cleaned and re-sterilized between batches. Cleaning
validation as well as process validation is relatively difficult
[87]. Additionally, the largest system available is the module
400, which has a surface area of 340 000 cm2 . The Cell
Factory possesses a number of attractive features, which
include unaltered growth kinetics from laboratory scale,
low risk of contamination and compact design. A number of
recent reports have shown the potential of the Cell Factory
for the production of vaccines and recombinant proteins
[89–92]. However, the largest size module available is the
40-tray system, which has a surface area of 25 280 cm2 . To
put these surface areas into perspective, a 12-litre working
volume of Cytodex microcarriers at a concentration of 5 g/l


C 2006 Portland Press Ltd
10 J. N. Warnock and M. Al-Rubeai
would have a greater surface area than the CellCube or the
Cell Factory.
The Wave bioreactor can be used to culture suspension
cells or microcarrier cultures and is marketed as an alter-
native to the traditional stainless-steel bioreactor. Cells are
cultured in pre-sterilized plastic bags (called Cellbags® ),
which are single-use. The bags are filled with medium and
cells and inflated to form a gas-permeable chamber. It is then
placed on a rocking platform, which allows for excellent
oxygenation and mixing with low shear stress. The largest
size currently available is 500 litres. Bag sizes of 1500–
2500 litres are becoming available, and HyClone reports
offering a 10 000-litre bag through its HyNetics option. The
performance of the Wave bioreactor compares favourably
with conventional cultivation vessels such as stirred-tank
bioreactors and roller cultures with respect to simplicity of
operation and cost [93]. They have been successfully used to
produce adeno-associated viruses from baculovirus/insect
cell culture [94], and adenovirus vectors using PER.C6 cells
[95,96].
Conclusion
During the past 40 years, several strategies have been
formulated to produce biopharmaceutical products from
animal cells. As the demand for such products increases over
the coming years, the efficiency of the production methods
will become even more important. The STR and the airlift
reactor have become industry standards, and, owing to their
ease of operation and ease of licensing, homogeneous reac-
tors will almost certainly remain at the forefront of animal
cell biotechnology. Furthermore, cell retention systems are
now enabling STRs to run in perfusion mode for ex-
tended duration, which increases their efficiency relative
to either batch or fed-batch cultures, which is the current
industry standard. Of all the separation techniques currently
available, the acoustic filter and the ATF filter have the best
potential, although the power requirements for large-scale
acoustics filters are an issue. The recent interest in these
devices also suggests that they may be set to become an
industrial standard. Fixed and fluidized-bed systems have
been used as high-density methods of culturing anchorage-
dependent cells. Results for CellCube disposable reactors
and packed-bed reactors have shown great promise, but
their suitability for commercial-scale production has been
brought into question. Therefore fluidized-bed bioreactors
may be a better option. The Wave bioreactor is currently
attracting a lot of attention, but there is a lack of published
information about its performance owing to its newness.
Initial studies do suggest that it has great potential for the
Good-Manufacturing-Practice manufacture of biopharma-
ceutical products. In the latest report by HighTech Business


C 2006 Portland Press Ltd
Decisions (Moraga, CA, U.S.A.), Biopharmaceutical Con-
tract Manufacturing 2005: Improved Processes and New
Capacity for Pipeline to Commercial Production (February
2005; http://www.hightechdecisions.com/), 51 directors of
biomanufacturing at pharmaceutical and biotechnology com-
panies worldwide were surveyed. More than half of these
directors are either using, or are interested in using, dis-
posable bags in-house or through a contract manufactur-
ing organization (http://www.contractpharma.com/articles/
2005/06/disposable-bioprocessing.php). Finally, choosing an
appropriate production method for any biologic will be of
paramount importance for its commercial success.
References
1 Anscomb, A. (2004) in Kalorama Information Market
Intelligence (Heffner, S., ed.), Kalorama Information, New York
2 Sinclair, A. (2004) in Advances in Large-scale Biopharma-
ceutical Manufacturing and Scale-up Production (Langer, E. S.,
ed.), ASM Press and the Institute for Science and Technology
Management, Washington, DC
3 Butler, M. (1987) Adv. Biochem. Eng. Biotechnol. 34,
57–84
4 Hu, W. S. and Peshwa, M. V. (1991) Can. J. Chem. Eng. 69,
409–420
5 van Wezel, A. L. (1967) Nature (London) 216, 64–65
6 Nikolai, T. J. and Hu, W. S. (1992) Enzyme Microb. Technol.
14, 203–208
7 Onderwater, R. C., Goeptar, A. R., Levering, P. R., Vos, R. M.,
Konings, P. N., Doehmer, J., Commandeur, J. N. and Vermeulen,
N. P. (1996) Protein Expression Purif. 8, 439–446
8 Warnock, J. N. and Al Rubeai, M. (2005) in Applications of
Cell Immobilisation Biotechnology (Nedovic, V. and Willaert,
R., eds.), pp. 423–438, Springer, Berlin
9 Berry, J. M., Barnabe, N., Coombs, K. M. and Butler, M. (1999)
Biotechnol. Bioeng. 62, 12–19
10 Chu, L. and Robinson, D. K. (2001) Curr. Opin. Biotechnol. 12,
180–187
11 Bailey, J. E. and Ollis, D. F. (1986) Biochemical Engineering
Fundamentals, 2nd edn, McGraw-Hill, Jurong, Singapore
12 Oh, S. K., Nienow, A. W., Al Rubeai, M. and Emery, A. N.
(1992) J. Biotechnol. 22, 245–270
13 Al Rubeai, M., Emery, A. N., Chalder, S. and Goldman, M. H.
(1993) J. Biotechnol. 31, 161–177
14 Kioukia, N., Nienow, A. W., Emery, A. N. and Al Rubeai, M.
(1992) Trans. Inst. Chem. Eng. 70C, 143–148
15 Hu, Y. C., Kaufman, J., Cho, M. W., Golding, H. and Shiloach, J.
(2000) Biotechnol. Prog. 16, 744–750
16 Kennard, M. L. and Piret, J. M. (1994) Biotechnol. Bioeng. 44,
45–54
17 Yamaji, H. and Fukuda, H. (1994) Appl. Microbiol. Biotechnol.
42, 531–535
 Bioreactors for cell culture 11

18 Kennard, M. L. and Piret, J. M. (1995) Biotechnol. Bioeng. 47, 43 Williams, S. N. O., Callies, R. M. and Brindle, K. M. (1997)
550–556 Biotechnol. Bioeng. 56, 56–61
19 Nikolai, T. J. and Hu, W. S. (1992) Enzyme Microb. Technol. 44 Koska, J., Bowen, B. D. and Piret, J. M. (1997) Chem. Eng. Sci.
14, 203–208 52, 2251–2263
20 Schweikart, F., Jones, R., Jaton, J.-C. and Hughes, G. J. (1999) 45 Fassnacht, D. and Portner, R. (1999) J. Biotechnol. 72, 169–184
J. Biotechnol. 69, 191–201 46 Glacken, M. W., Fleischaker, R. J. and Sinskey, A. J. (1983)
21 Xiao, C., Huang, Z., Li, W., Hu, X., Qu, W., Gao, L. and Liu, G. Ann. N.Y. Acad. Sci. 413, 355–373
(1999) Cytotechnology 30, 143–147 47 Knight, P. (1989) Bio/Technology 7, 459–461
22 Yamaji, H. and Fukuda, H. (1997) J. Ferment. Bioeng. 83, 48 Bratch, K. and Al Rubeai, M. (2001) Biotechnol. Lett. 23,
489–491 137–141
23 Cosgrove, L., Lovrecz, G. O., Verkuylen, A., Cavaleri, L., Black, 49 Shirgaonkar, I. Z., Lanthier, S. and Kamen, A. (2004) Biotechnol.
L. A., Bentley, J. D., Howlett, G. J., Gray, P. P., Ward, C. W. and Adv. 22, 433–444
McKern, N. M. (1995) Protein Expression Purif. 6, 789–798 50 Dalm, M. C., Jansen, M., Keijzer, T. M., van Grunsven, W. M.,
24 Crowley, J., Marsden, W. L. and Gray, P. P. (1991) in Animal Cell Oudshoorn, A., Tramper, J. and Martens, D. E. (2005)
Culture and Production of Biologicals (Sasaki, R. and Ikura, H., Biotechnol. Bioeng. 91, 894–900
eds.), pp. 275–281, Kluwer Academic, Dordrecht 51 Pui, P. W., Trampler, F., Sonderhoff, S. A., Groeschl, M., Kilburn,
25 Hesse, F., Ebel, M., Konisch, N., Sterlinski, R., Kessler, W. and D. G. and Piret, J. M. (1995) Biotechnol. Prog. 11, 146–152
Wagner, R. (2003) Biotechnol. Prog. 19, 833–843 52 Trampler, F., Sonderhoff, S. A., Pui, P. W., Kilburn, D. G. and
26 Merchuk, J. C. (2003) Can. J. Chem. Eng. 81, 324–337 Piret, J. M. (1994) Bio/Technology 12, 281–284
27 Kadouri, A. and Spier, R. E. (1997) Cytotechnology 24, 89–98 53 Coakley, W. T., Whitworth, G., Grundy, M. A., Gould, R. K. and
28 Voisard, D., Meuwly, F., Ruffieux, P. A., Baer, G. and Kadouri, A. Allman, R. (1994) Bioseparation 4, 73–83
(2003) Biotechnol. Bioeng. 82, 751–765 54 Gorenflo, V. M., Smith, L., Dedinsky, B., Persson, B. and Piret,
29 Deo, Y. M., Mahadevan, M. D. and Fuchs, R. (1996) J. M. (2002) Biotechnol. Bioeng. 80, 438–444
Biotechnol. Prog. 12, 57–64 55 Dalm, M. C., Cuijten, S. M., van Grunsven, W. M., Tramper,
30 Iding, K., Lütkmeyer, D., Fraune, E., Gerlach, K. and Lehmann, J. J. and Martens, D. E. (2004) Biotechnol. Bioeng. 88,
(2000) Cytotechnology 34, 141–151 547–557
31 van Wezel, A. L., van der Velden-de Groot, C. A. M., de 56 Gorenflo, V. M., Angepat, S., Bowen, B. D. and Piret, J. M.
Haan, H. H., van den Heuvan, N. and Schasfoort, R. (1985) (2003) Biotechnol. Prog. 19, 30–36
Dev. Biol. Stand. 60, 229–236 57 Merten, O. W. (2000) Cytotechnology 34, 175–179
32 Avgerinos, G. C., Drapeau, D., Socolow, J. S., Mao, J. I., Hsiao, 58 Dalm, M. C., Jansen, M., Keijzer, T. M., van Grunsven, W. M.,
K. and Broeze, R. J. (1990) Bio/Technology 8, 54–58 Oudshoorn, A., Tramper, J. and Martens, D. E. (2005)
33 McLimans, W. F., Kwasniewski, B., Robinson, F. O., Chu, T. M., Biotechnol. Bioeng. 91, 894–900
Sufrin, G. and Gailani, S. (1977) Cancer Treat. Rep. 61, 59 de la Broise, D., Noiseux, M. and Lemieux, R. (1991)
161–165 Biotechnol. Bioeng. 38, 781–787
34 Jan, D. C., Emery, A. N. and Al Rubeai, M. (1993) Biotechnol. 60 de la Broise, D., Noiseux, M., Massie, B. and Lemieux, R. (1992)
Tech. 7, 351–356 Biotechnol. Bioeng. 40, 25–32
35 Bierau, H., Perani, A., Al Rubeai, M. and Emery, A. N. (1998) 61 Kawahara, H., Mitsuda, S., Kumazawa, E. and Takeshita, Y.
J. Biotechnol. 62, 195–207 (1994) Cytotechnology 24, 89–98
36 Jan, D. C., Jones, S. J., Emery, A. N. and Al Rubeai, M. (1994) 62 Bleckwenn, N. A., Golding, H., Bentley, W. E. and Shiloach, J.
Cytotechnology 16, 17–26 (2005) Biotechnol. Bioeng. 90, 663–674
37 Amos, B., Al Rubeai, M. and Emery, A. N. (1994) Enzyme 63 Kadouri, A. and Zipori, D. (1989) in Advances in Animal Cell
Microb. Technol. 16, 688–695 Biology and Technology for Bioprocesses (Spier, R. E., Griffiths,
38 Adamson, S., Fitzpatrick, S., Behie, L. A., Gaucher, G. and J. B., Stephenne, J. and Grooy, P. J., eds.), pp. 327–330, Courier
Lesser, B. (1983) Biotechnol. Lett. 5, 573–578 International, Tiptree, Essex, U.K.
39 Sjorgren-Jansson, E. and Jeansson, S. (1985) J. Immunol. 64 Kang, S.-H., Lee, G. M. and Kim, B.-G. (2000) Cytotechnology
Methods 84, 359–364 34, 151–158
40 Comer, M. J., Kearns, M. J., Wahl, J., Munster, M., Lorenz, T., 65 Kaufman, J., Wang, G., Zhang, W., Valle, M. A. and Shiloach, J.
Szperalski, B., Koch, S., Behrendt, U. and Brunner, H. (1990) (2000) Cytotechnology 33, 3–11
Cytotechnology 3, 295–299 66 Merten, O. W., Cruz, P. E., Rochette, C., Geny-Fiamma, C.,
41 Kessler, N., Thomas, G., Gerentes, L., Delfosse, G. and Aymard, Bouquet, C., Goncalves, D., Danos, O. and Carrondo, M. J.
M. (1997) Cytotechnology 24, 109–119 (2001) Biotechnol. Prog. 17, 326–335
42 Thelwell, P. E. and Brindle, K. M. (1999) Cytotechnology 30, 67 Wang, G., Zhang, W., Jacklin, C., Freedman, D., Eppstein, L.
121–132 and Kadouri, A. (1992) Cytotechnology 9, 41–49

C 2006 Portland Press Ltd
12 J. N. Warnock and M. Al-Rubeai

68 Park, S. and Stephanopoulos, G. (1993) Biotechnol. Bioeng. 85 Warnock, J. N., Price, T., Slade, A. and Al Rubeai, M. (2004)
41, 25–34 Bioprocess. J. 3, 41–45
69 Chiou, T. W., Murakami, S. and Wang, D. I. C. (1991) 86 Kotani, H., Newton, III, P. B., Zhang, S., Chiang, Y. L., Otto, E.,
Biotechnol. Bioeng. 37, 755–761 Weaver, L., Blaese, R. M., Anderson, W. F. and McGarrity, G. J.
70 Park, S. and Stephanopoulos, G. (1993) Biotechnol. Bioeng. (1994) Hum. Gene Ther. 5, 19–28
41, 25–34 87 Wikström, K., Blomberg, P. and Islam, K. B. (2004)
71 McTaggart, S. and Al Rubeai, M. (2000) Biotechnol. Prog. 16, Biotechnol. Prog. 20, 1198–1203
859–865 88 Merten, O. W. (2004) J. Gene Med. 6 (Suppl. 1), S105–S124
72 Warnock, J. N. (2002) Ph.D. Thesis, University of Birmingham 89 Davis-Sproul, J. M., Harris, M. P., Davidson, N. E., Kobrin, B. J.,
73 Kafri, T. (2001) Curr. Opin. Mol. Ther. 3, 316–326 Jaffee, E. M. and Emens, L. A. (2005) Cytotherapy 7, 46–56
74 Shi, Y., Ryu, D. D. Y. and Park, S. (1992) Biotechnol. Bioeng. 40, 90 Ho, L., Greene, C. L., Schmidt, A. W. and Huang, L. H. (2004)
260–270 Cytotechnology 45, 117–123
75 Rodrigues, M. T. A., Vilaça, P. R., Garbuio, A., Takagi, M., Barbosa, 91 Ni, Y. J., Sun, S. S., Oparaocha, T., Humeau, L., Davis, B.,
Jr, S., Léo, P., Laignier, N. S., Silva, A. A. P. and Moro, A. M. (1999) Cohen, R., Binder, G., Chang, Y. N., Slepushkin, V. and Dropulic,
Bioprocess Eng. 21, 543–546 B. (2005) J. Gene Med. 7, 818–834
76 Portner, R., Bohmann, A., Ludemann, I. and Markl, H. (1994) 92 Tuyaerts, S., Noppe, S. M., Corthals, J., Breckpot, K., Heirman,
J. Biotechnol. 34, 237–246 C., De Greef, C., Van Riet, I. and Thielemans, K. (2002) J.
77 Kratje, R. B. and Wagner, R. (1992) Biotechnol. Bioeng. 39, Immunol. Methods 264, 135–151
233–242 93 Weber, W., Weber, E., Geisse, S. and Memmert, K. (2002)
78 Preissmann, A., Wiesmann, R., Buchholz, R., Werner, R. G. and Cytotechnology 38, 77–85
Noe, W. (1997) Cytotechnology 24, 121–134 94 Chen, H. F., Zhou, S. Z., Pierce, G. P. and Colosi, P. (2004)
79 Kratje, R. B., Reimann, A., Hammer, J. and Wagner, R. (1994) Mol. Ther. 9, S160–S160
Biotechnol. Prog. 10, 410–420 95 Ekstrom, D., Cheng, W., Andersson, R., Mitra, G. and Zhu, J. W.
80 Frame, K. K. and Hu, W. S. (1990) Biotechnol. Bioeng. 35, (2004) Abstracts of Papers of the American Chemical Society
469–476 227, U202
81 Leelavatcharamas, V., Emery, A. N. and Al Rubeai, M. (1994) 96 Mikola, M. R., Burden, E. C., Jug-Dujakovic, M., Pearre, C. L.,
Cytotechnology 15, 65–71 Herber, W. K. and Amanullah, A. (2004) Abstracts of Papers
82 Goldman, M. H., James, D. C., Rendall, M., Ison, A. P., of the American Chemical Society 227, U222
Hoare, M. and Bull, A. T. (1998) Biotechnol. Bioeng. 60, 97 Singh, R. P., Al Rubeai, M., Gregory, C. D. and Emery, A. N.
596–607 (1994) Biotechnol. Bioeng. 44, 720–726
83 Wang, M. D., Yang, M., Huzel, N. and Butler, M. (2002)
Biotechnol. Bioeng. 77, 194–203 Received 6 December 2005/21 March 2006; accepted 28 March 2006
84 Kong, D., Cardak, S., Chen, M., Gentz, R. and Zhang, J. (1999) Published on the Internet 20 June 2006, doi:10.1042/BA20050233
Cytotechnology 29, 215–220

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