MAMMALIAN CELL BIOREACTORS
and other culture conditions at very large scale (over
WEICHANG ZHOU1 , GARGI
SETH2 , MARIA J. GUARDIA3 ,
and WEI-SHOU HU4
1 Genzyme Corporation,
Framingham,
Massachusetts
2 Genentech, Inc., 1 DNA Way,
South San Francisco,
California
3 Camino De Purchill, Puleva
Biotech Department of
Process Engineering,
Granada, Spain
4 University of Minnesota,
Department of Chemical
Engineering and Materials
Science, Minneapolis,
Minnesota
INTRODUCTION
The year 2007 marked the 20th anniversary of the intro-
duction of the first recombinant mammalian cell culture
produced therapeutic protein, tissue plasminogen activa-
tor (Activase) by Genentech. The first Chinese hamster
ovary (CHO) cell produced protein drug was followed by
the introduction of erythropoietin (EPO) (by Amgen in
1989). In the past decade, a rapid expansion of recombi-
nant antibody-based therapeutics has pushed the annual
production amount of therapeutic protein to tens of thou-
sands of kilograms and over 20 billion US dollars in
revenue per annum (Tables 1 and 2). Before the advent of
recombinant DNA technology, industrial mammalian cell
culture was used primarily for the production of biologics
using isolated transformed cells and viral vaccines using
primary cells and normal diploid cell strains. The arrival
of rDNA technology and the expression of heterologous
proteins in mammalian cells completely transformed cell
culture technology. Except for the production of attenuated
or inactivated viral vaccines, virtually all mammalian cell
culture processes now employ continuous cell lines, most of
which have been adapted to grow in suspension, without
a surface support. The adoption of the growth of sus-
pension cells as the typical vehicle for the production of
complex molecules requiring extensive posttranslational
modification also transformed the concept of bioreactor
selection.
Most research and many commercial efforts on devel-
oping bioreactors for mammalian cell culture occurred in
the 1980s and early 1990s. The anticipation was that the
potentially detrimental effect of the air sparging on mam-
malian cells would require a newly designed bioreactor to
accommodate the manufacturing needs of the new mam-
malian cell-derived products. What was unanticipated to
researchers and altered the landscape of bioprocessing
technology was the extraordinary capability of cells to
Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, edited by Michael C. Flickinger
Copyright 2010 John Wiley & Sons, Inc.
adapt to suspension growth, serum-free, even protein-free,
20,000 L). Over time the bioreactors used for industrial
cell culture converge on a few basic configurations. This
chapter focuses only on the main types used currently
for industrial cell culture. It should be noted that many
of the bioreactors developed in the 1980s, although not
commonly used, might find specialized applications in
other areas, such as tissue engineering, gene therapy, and
cellular therapy.
BASIC REACTOR OPERATION AND KINETICS
Basic Types of Reactors
Mammalian cell bioreactors can be generally categorized
similarly to chemical reactors according to their mix-
ing characteristics. It is instructive to review two ideal
reactors: well-mixed stirred-tank- and plug-flow (tubular)
reactor. In an ideal well-mixed bioreactor, the mixing is
assumed to be intense enough that the fluid is homoge-
neous through the reactor. The mathematical description
of ideal continuous flow stirred-tank reactor is described
by the following first-order differential equation.
d(VCA )
= Fi CAi Fo CAo + rV (1)
dt A
V is the culture volume in the bioreactor, CA the concen-
tration of nutrient or product A, t is time, F is the flow
rate, and rA is the volumetric consumption rate of nutrient
or production rate of product A.
In an ideal continuous stirred-tank reactor, there is no
flow bypass and no shunt of substrate from inlet to outlet,
no dead zones or clumps of undissolved solid substrate
floating around. Any substrate added through feeding is
instantaneously distributed throughout the entire reactor,
and when gas sparging is employed, the agitator provides
an intimately mixed gasliquid.
The basic model for the tubular reactor (such as hollow
fiber and many other membrane bioreactor systems that
are described later in this chapter) is that liquid phase
moves as a plug-flow, meaning that there is neither varia-
tion of axial velocity over the cross section nor backmixing
in the longitudinal direction. The mass balance for com-
ponent A in a volume element Sz that described an ideal
plug-flow reactor is as follows:
CA CA
= vZ S + rA (2)
t z
where vz is the linear velocity in the z direction and S is
the cross-sectional area.
Given the initial condition in Equation 1, or both initial
and boundary condition in Equation 2, one can obtain the
concentration profiles over time; or for tubular reactors,
the profile can be obtained over time and spatial posi-
tion. At steady state (i.e. cell concentration and cellular
activities are not changing with time), the equation for
tubular reaction becomes
1
2 MAMMALIAN CELL BIOREACTORS

Table 1. Major Licensed Recombinant Therapeutic Proteins Expressed by Mammalian Cells

Protein Cell Line Used Therapeutic Use (Year Approved)

Tissue plasminogen activator (TPA) CHO Heart attack/embolism, stroke (1987)
Erythropoietin (EPO) CHO Anemia (1989)
Human growth hormone C127 Growth deficiency, renal insufficiency (1989)
Granulocyte colony stimulating factor CHO Neutropenia (1991)
Factor VIII CHO/BHK-2 Hemophilia A (1992 & 1993)
Deoxyribonuclease I (DNase) CHO Cystic fibrosis (1993)
Glucocerebrosidase CHO Gauchers disease (1994)
Interferon- CHO Multiple sclerosis (1996)
Factor IX CHO Hemophilia B (1997)

Table 2. Licensed Therapeutic Monoclonal Antibodies and Antibody Fusion Proteins in the United States

Protein (Trade Name) Cell Line Used Therapeutic Use (Year Approved)
Anti-CD3 (Orthoclone OKT3) Hybridoma Allograft rejection (1986)
Anti-GPIIb/IIIa Fab fragment (ReoPro), Sp2/0 Blood clots in angioplasty (1994)
Anti-IL 2 receptor a chain (CD25) (Zenapax) NS0 Renal transplantation (1997)
Anti-CD20 (Rituxan) CHO Non-Hodgkins lymphoma (1997)
Anti-IL 2 receptor a chain (CD25) (Simulect) Sp2/0 Renal transplantation (1998)
Anti-respiratory syncytial virus (Synagis) NS0 Respiratory syncytial virus (1998)
Anti-tumor necrosis factor a (Remicade) Sp2/0 Crohns disease (1998)
Anti-HER2 (Herceptin) CHO Metastatic breast cancer (1998)
Anti-tumor necrosis factor Fc fusion (Enbrel) CHO Rheumatoid arthritis (1998)
Anti-CD33 (Mylotarg) NS0 Acute myeloid leukemia (2000)
Anti-CD52 (Campath) CHO B cell chronic lymphocytic leukemia (2001)
Anti-CD20 (Zevalin) CHO Non-Hodgkins lymphoma (2002)
Anti-tumor necrosis factor a (Humira) CHO Rheumatoid arthritis (2002)
Anti-IgE (Xolair) CHO Asthma (2003)
Anti-CD20 (Bexxar) Hybridoma Non-Hodgkins lymphoma (2003)
Anti-leukocyte function antigen-1 (Raptiva) CHO Psoriasis (2003)
Anti-leukocyte function antigen-3 (CD2) Fc fusion CHO Psoriasis (2003)
(Amevive)
Anti-epidermal growth factor receptor (Erbitux) Sp2/0 Colorectal cancer (2004)
Anti-vascular endothelial growth factor (Avastin) CHO Colorectal cancer (2004)
Anti-alpha4-subunit of alpha4beta1 and alpha4beta7 NS0 Multiple sclerosis (2004)
integrins (Tysabri)
Anti-CD80 and CD86 (Orencia), Fc fusion CHO Rheumatoid arthritis (2005)
Anti-epidermal growth factor receptor (Vectibix) CHO Metastatic colorectal cancer (2006)
Anti-C5 (the complement protein) (Soliris) NS0 Paroxysmal nocturnal hemoglobinuria (PNH) (2007)

CA rA S in the reactor. The other extreme of mixing is total seg-

= (3)
z F
which describes changes of concentration of A along the
direction of fluid flow. It is clear that the nutrient con-
centration will decrease from inlet to the distal end of
the reactor, while metabolite or product concentration
increases. The length of the reactor is limited because
eventually nutrient depletion or metabolite accumulation
inhibits growth and metabolism.
These ideal cases of well-mixed tanks or plug-flow tubu-
lar reactors are situations that can be approximated in
small-scale laboratory conditions. The conditions in larger
scale reactors deviate significantly from these ideal condi-
tions.
In a well-mixed bioreactor, there are no concentration
gradients in either the gas or the liquid phase. In other
words, none of the chemical species or cells is segregated
regation where there is no interaction between different
volume elements in the bioreactor. An ideal plug-flow reac-
tor is assumed to be under conditions of total segregation.
Most bioreactor systems have a mixing pattern between
the two extremes and are under partially segregated con-
ditions.
In general, laboratory and small pilot plant bioreactors
are used for process development, optimization, and char-
acterization. The fluid mixing characteristics are rather
sensitive to the scale of the reactor. Furthermore, plug-flow
bioreactors are intrinsically more difficult to scale up than
mixing vessels, as the concentration gradient of essen-
tial nutrients, oxygen in particular, will inevitably become
limiting in the downstream region of the reactor. In con-
sidering the selection of bioreactors for mammalian cell
cultures, the mixing characteristics and their relationship
to scale-up have to be kept in mind.

Batch/Fed-batch Versus Continuous/Perfusion Operations
Mammalian cell cultures can be operated in batch,
fed-batch, and continuous modes. In a batch culture all
nutrients are added at the beginning of a production cycle,
except that oxygen is supplied continuously by diffusion
through the interface with an oxygen carrying stream,
which is part of the recirculating medium or air flowing
through the culture. Using a conventional medium,
the typical concentrations achieved in a batch culture
are rather low, around 2 4 106 cells/mL. In many
cases, batch operation tends to be run as extended batch
processes that are sometimes referred to as repeated batch
cultures with intermittent harvest. In this operation mode,
spent medium with or without cells may be harvested
periodically and replaced by an equal quantity of fresh
medium. It allows a production process to be extended
to a few weeks with an increased productivity for the
reactor. With this technique it is possible to prevent the
inhibitory products from accumulating to an exceedingly
high level and to provide nutrients for cell growth and
production.
To prolong the culture period, fed-batch operation is
practised. The culture is started at less than full vol-
ume; after a short period of growth, concentrated nutrient
stream containing amino acids, glucose, and some other
nutrients is added continuously or intermittently until
reaching maximum volume. The total amount of nutri-
ents added to the reactor is typically substantially (a
few fold) higher than if the reactor was filled with fresh
basal medium at the beginning. Such fed-batch processes
reach higher cell concentrations and prolong the prod-
uct formation period. The final product concentration is
substantially higher than that in a batch culture.
Continuous chemostat is a very useful research tool.
Once a steady state is reached, the spent medium with-
drawn is balanced by equal supply rate of fresh medium,
and the amount of cells being continuously discharged
from the bioreactor is balanced by growth. A drawback
of a simple continuous culture is the low cell concentra-
tion, and the corresponding low product throughput of the
system. To increase the cell concentration and the reactor
throughput, a perfusion culture is more frequently used in
industrial continuous operations. In a perfusion culture,
a high medium flow rate (one much higher than the cell
growth rate, which would wash out cells in a simple con-
tinuous culture) is used, but a cell retention device is used
to prevent most of the cells from leaving the reactor. The
cell concentration in the reactor is many times higher than
that for a simple continuous culture.
A cell retention device may be physically positioned
inside or outside of the bioreactor vessel (1,2). The devices
that have been used include settling tanks (3), external
centrifuges (4) (5), external membrane filtration (6), inter-
nal microfiltration (7), rotating wire cage (spin filters)
(8), and acoustic settling device (9). All these, or similar
devices, have been shown to be effective at laboratory scale.
On the industrial scale, bioreactors of up to hundreds of
liters, centrifuges, settling devices, and spin filters appear
to be the top choices. The employment of a centrifuge or a
settling device necessitates a recirculation loop to return
MAMMALIAN CELL BIOREACTORS 3
the concentrated cell stream back to the reactor, while
discarding the purged cells along with the other stream.
As the scale increases the holding time in the external
recirculating loop also becomes longer and potential oxy-
gen starvation must be avoided to minimize the risk of cell
progression to apoptosis. Such concern may require the
reduction of temperature in the external loop. Using an
internal cell retention device minimizes such a concern.
However, all cell retention devices may face occasional
problems of cell clogging or mechanical failure. An exter-
nal device is in general easier to be replaced or duplicated
for alternating operation than an internal device. When a
culture is operated in a continuous mode, it is desirable
for the operation to last for months; a replaceable cell
retention device can make the process more robust.
CELL SUPPORT FOR MIXING VESSELS
Microcarriers
The use of microcarriers for cell culture was first demon-
strated four decades ago (10). The basic concept is to
allow cells to attach to the surface of small suspended
beads so that conventional stirred-tank bioreactors can
be used for cell cultivation. The diameter and density of
these cell-laden microcarriers are usually in the range
of 100300 m and 1.021.05 g/cm3 so that the settling
velocity is in the order of centimeter per minute. This
diameter range also gives a good growth surface area
per reactor volume. Even at a moderate microcarrier con-
centration, in the range of 815% culture volume being
occupied by microcarriers, a significantly larger surface
area per reactor volume can be achieved in a microcarrier
culture than in T-flasks, roller bottles, or other plastic
reactors with flat surfaces.
Although even smaller microcarriers, less than
100 m in diameter, can provide more surface area, they
are not used often. Most adherent-dependent cells do not
develop their normal morphology, and in many cases,
do not multiply well on surfaces with an excessively
high curvature. These cells do not grow well on small
microcarriers. On the other hand, some cells, especially
transformed cells, multiply well even though they are
attached to small microcarriers and do not spread.
These cells, after attaching to the small microcarriers,
agglomerate to form aggregates and continue to grow to
high density. The small microcarriers, usually with a
diameter of about 50 m, serve as nuclei for the initiation
of aggregate formation (11).
The microcarriers can be made of many different mate-
rials, including dextran, gelatin (12), polystyrene (13,14),
glass (15), and cellulose (16). Not all these are com-
mercially available. In general, in addition to having a
wettable surface, the backbone materials of microcar-
riers often need to be chemically modified to improve
cell attachment. One of the most widely used microcarri-
ers, the dextran based ones, are derivatized with charged
molecules or collagen. Polystyrene microcarriers have also
been coated with collagen or other adhesion molecules for
better performance.
4 MAMMALIAN CELL BIOREACTORS
An advantage of microcarriers for the industrial-scale
culture of anchorage-dependent cells is the ease of
separating cells from culture medium. Many microcarrier
cultures require medium exchanges during cultivation
to remove accumulating lactate, ammonia, and other
metabolites and to replenish nutrients. In many cases,
the cell-laden microcarriers are simply allowed to settle
and the spent medium is withdrawn and replenished.
In large-scale operations, continuous or semicontin-
uous perfusion is more frequently used. This can be
accomplished by withdrawing medium through a coarse
screen that allows medium to pass through but retains
microcarriers in the reactor. Many types of cells have been
grown on microcarriers, including fibroblasts, kidney,
epithelial, hepatocyte, neuroblastoma, and endothelial
cells from various species. Overall, microcarrier culture is
a convenient laboratory and research tool, and it has the
advantage of being amenable to large-scale production if
a large quantity of product is needed.
Macroporous Microcarriers
A variant of microcarriers is one with large pores of tens
of micrometers in its interior. The void space inside allows
cells to be cultivated not only on the external surface but
also internally. Cells in the interior are less susceptible to
mechanical damage caused by agitation and gas sparging.
Of course, being in the interior of microcarriers, cells are
more likely to be subject to oxygen limitation due to the
long diffusional distance, especially since most macrop-
orous microcarriers have a larger diameter (500 m to
a couple of millimeters). Macroporous microcarriers are
made of different materials, including gelatin (17), colla-
gen (18), and plastic (e.g. Cytoline). Some macroporous
microcarriers have apparent bulk density similar to that
of conventional microcarriers. However, because of their
larger particle size, the settling velocity is substantially
higher. In some cases, the settling velocity is orders of
magnitude higher, in the order 1001000 cm/min. These
microcarriers are suitable for use in fluidized beds with
a high fluid recirculation rate. Because of the high set-
tling velocity even at high upward supervelocity, the
carrier can be retained in the reactor. Similar to micro-
carriers many cell lines have been successfully grown on
macroporous microcarriers including Vero, HepG2, CHO,
and HEK293 cells. The final cell concentration achieved
tends to be higher than that obtained with an equivalent
volumetric concentration of conventional microcarriers.
But, in some cases, the growth kinetics are a bit slower
because the penetration of cells into the interior may be
slowed or even retarded by restrictive opening of these
pores.
Since the cells are protected inside the capsules, they
are protected from hydrodynamic damage. The culture
can be stirred at faster rates than conventional culture,
which improves nutrient, metabolite, and gas exchange.
Entrapped cells may be cultured in stirred-tank bioreac-
tors, fixed-bed and fluidized-bed reactors. An airlift reactor
could be used as well, provided that the beads are small
and not significantly denser than the medium.
Aggregates
Some transformed cells that normally attach to or spread
and grow on a surface can form aggregates when culti-
vated on less hydrophilic surfaces or in shaker flasks or
stirred vessels (19). Different methods have been used to
induce aggregate formation for cells. Aggregation can be
promoted by manipulating the calcium concentration (20)
in conjunction with the agitation rate. Aggregate cultures
have advantages similar to microcarrier cultures. They
can be cultivated in conventional stirred-tank reactors
with environmental control. They can be allowed to set-
tle relatively rapidly by stopping agitation. This permits
medium replenishment readily.
For many cell types, one limitation for application of
the aggregate culture is that the rate of aggregate for-
mation is slow. One way to induce aggregate formation
is to add microspheres, the small microcarriers with a
diameter of about 50 m, to cell suspension to allow for
rapid agglomeration to the microspheres (11). If the aggre-
gate diameters become too large, necrotic centers can be
formed due to transport limitation. The aggregate size may
influence the viral infection kinetic and yield for vaccine
formation (21). Many cell lines, including BHK, CHO, 293,
and ST cells, have been cultivated as aggregates with sizes
ranging between 90 and 400 m, without the formation of
necrotic centers (11,22).
Cell Entrapment
Another method of cell cultivation, which enables the
use of a stirred-tank reactor, is cell entrapment. This
technique entails entrapping cells in polymeric spheres.
The spheres can then be coated with another polymeric
film with controlled cross-linking of the polymer to mod-
ulate the permeability to molecule according to their
size. This is commonly referred to as microencapsula-
tion. In other applications, the spheres with entrapped
cells are cultivated in the same way as the beads sus-
pended in medium to allow for cell growth. One of the
polymers frequently used for cell entrapment is calcium
alginate. Cell entrapment in calcium alginate is accom-
plished by preparing a cell suspension in sodium alginate
and adding it, in a drop-wise fashion, into a solution
of calcium chloride. A high calcium concentration pro-
motes cross-linking of alginate, instantaneously forming
beads containing entrapped cells. The alginate beads may
be coated with polylysine for increased mechanical char-
acteristics and structural integrity, but such treatment
decreases the molecular weight cutoff (MWCO) and pro-
hibits large-molecular weight proteins in the medium
from reaching the cells. To prepare hollow spheres, the
alginate gel inside a bead coated with a polylysine is liq-
uefied by treatment with a calcium chelator such as EDTA
(ethylenediaminetetraacetic acid) or citrate.
The diameter of these beads is often in the order of
millimeters. The mechanical strength of the gel which con-
stitutes the beads is relatively weak even with polymeric
coating. Large-scale application using such techniques is
not easy. On the other hand, the microcapsule provides
a means of immunoisolation of transplanted cells or tis-
sues (sometimes referred to as artificial cells) and could
 MAMMALIAN CELL BIOREACTORS 5

be suitable for some tissue engineering applications. Cell

entrapment has been applied to hybridomas (23) and small
clusters of adherent cells (24). It has also found appli-
cations in the cultivation of differentiated cells, such as
insulin-secreting cells. For tissue engineering applications
using differentiated cells, the enclosing membrane around
the cells and the hydrogel must be biocompatible (25).

CELL CULTURE BIOREACTORS

Cells respond to fluid flow by changing their morphology,

metabolism, and, in some cases, their signaling pathways.
Excessive fluid shear causes cell damage and ultimately
death. How cells respond to fluid flow is dependent on cell
types, as well as the way they are cultivated. For cells
capable of growing either in suspension or attached, the
sensitivity to fluid flow may differ when grown in suspen-
sion or on a surface. Adherent cells may respond differently
on a flat surface and on a curved surface such as microcar-
riers. Even surface roughness or adhesiveness may affect
how they respond to fluid flow field. During the reactor
selection process, the most fundamental consideration is
that the reactor provides sufficient mixing and mass trans-
fer while minimizing potential cell damage. Cell damage
from the interaction between microcarriers and turbulent
eddies may be severe. Thus, fluid shear is of paramount
consideration in microcarrier culture. This is especially of
concern when the Kolmogorov length scale for the small-
est turbulent eddies becomes comparable to the diameter
of the microcarriers (26). The microcarriermicrocarrier
collisions are also expected to increase in frequency and
severity in the presence of eddies of size equal to the
average interparticle distance. Suspended cells are gen-
erally less prone to hydrodynamic damage than cells on
microcarriers. Overall, stirred-tank bioreactors are most
commonly used for cell cultivation. The use of disposable
cell culture devices (also referred to as a single-use device)
suitable for process scale (up to hundreds of liters) has
become common place in the past few years, especially in
cell propagation for inoculum preparation.
Stirred-Tank Bioreactor
Stirred tanks have been widely used for culturing sus-
pension cells since the 1960s. The basic configuration of
stirred-tank bioreactors for mammalian cell culture is
rather similar to that of microbial fermentors (Fig. 1). The
aspect ratio (the height-to-diameter ratio) of bioreactors
used for mammalian cell culture is often smaller than
that for microbial cultivation. However, in recent years
that distinction has become less apparent as the scale of
cell culture bioreactors approaches tens of cubic meters
in volume. However, the bioreactors used for microcarrier
cultures generally have a low aspect ratio to provide a
higher surface-to-volume ratio. The power input per unit
volume of bioreactor is substantially lower in mammalian
cell culture bioreactors. While the Rushton type impeller is
the norm in microbial fermentors, mammalian cell culture
bioreactors mostly employ pitched blade or marine type
impeller. Like in microbial fermentors, multiple impellers
are used in larger scale bioreactors for mammalian cell
Figure 1. Schematic of a stirred-tank bioreactor. (Courtesy of
Genentech, Inc.)
culture. The use of pitched blade, as opposed to Rushton
turbine, reflects the different purposes of agitation. In
microbial fermentation, agitation is needed at a higher
power input to disperse air bubbles and to increase oxygen
transfer efficiency, whereas in mammalian cell culture,
a much lower agitation rate is sufficient to keep cells or
microcarriers suspended in medium relatively uniformly
and to support the oxygen demand of the culture. In gen-
eral, the acceptable mixing time in a mammalian cell
culture bioreactor is substantially higher than that in a
microbial fermentor. The oxygen transfer capacity in a cell
culture bioreactor is substantially lower than that in a
microbial fermentor, since the typical oxygen demand in a
mammalian cell culture is also much lower (1050 times)
due to lower cell mass than that in microbial fermentation.
The most efficient way to supply oxygen, especially in
large scale, is the direct sparging of air or oxygen-enriched
air. Direct sparging, however, can potentially damage
cells. Numerous studies have tried to clarify the mech-
anism of cell damage caused by direct gas sparging (27).
The high energy released as the gas bubbles release from
the liquid surface due to bubble ruptures appears to be
responsible for its most damaging effects. These conse-
quences can be partially alleviated by the addition of
polyols in the culture medium to prevent cells from attach-
ing to the bubble surface. It is, in fact, standard practice
to include Pluronic F-68 in low protein or protein-free
medium for suspension cultures. This effectively prevents
cells from attaching to the bubble surface, thus minimizing
cell damage or death due to bubble ruptures. Commonly
used spargers include open pipe, ring sparger with multi-
ple holes of a diameter of 1/16 in., and microsparger made
6 MAMMALIAN CELL BIOREACTORS
of sintered stainless steel. Microspargers create swarms
of small bubbles and are efficient for oxygen transfer, as
they create a larger interfacial area at a given air flow
rate. With the use of microspargers, the gas flow rate
required to supply oxygen to maintain certain dissolved
oxygen levels tends to be smaller, leading to a lower CO2
removal rate and a generally higher CO2 concentration in
culture. Furthermore, in larger vessels, bubble coalescence
is unavoidable, thus offsetting the advantage of small bub-
bles. The open pipe or multiple-hole spargers are simple
and are generally used in large reactors. In general, gas
flow rates for sparging can be scaled up by keeping the
same superficial gas velocity. However, it is important to
increase the number of holes for sparging linearly to the
gas flow rates in order to keep the gas entrance velocity
at the same levels as it has been reported recently that a
high gas entrance velocity of more than 30 m/s may exert
an adverse effect on NS0 cells (28).
Airlift Bioreactor
An airlift reactor is essentially a relatively high aspect
ratio (tall and thin) tank with an internal concentric draft
tube as a guide for fluid recirculation (Fig. 2). The internal
liquid circulation is achieved by sparging at the bottom of
the draft tube. The sparged section has a lower effective
density than the bubble-free section, and the difference
in hydrostatic pressure between the two sections induces
the liquid circulation upward in the sparged section (riser)
and downward in the bubble-free section (downcomer). The
loop has the advantages of permitting high efficiency mass
transfer and improving the flow and mixing properties in
the vessel. A potential advantage of airlift reactors is the
low capital costs because of their simple mechanical con-
figuration. Although simple in construction, sound design
is critical for optimal hydrodynamic behavior. Airlift reac-
tors have been used successfully with suspension cultures
Air
Figure 2. Schematic of an airlift bioreactor.
of BHK 21, human lymphoblastoid, CHO, hybridomas,
and insect cells. In the past decade, the cell concentration
achieved in cell culture bioreactor increased substantially
as fed-batch culture became the prevailing mode of opera-
tion. In a fed-batch mode, the culture is often initiated at
about two thirds of the maximum volume. The other one
third is gradually fed throughout the cultivation period.
The initially low volume makes the use of airlift biore-
actors impractical as the draft tube is not completely
submerged in the medium. Furthermore, the increased
oxygen consumption caused by the high cell concentration
requires a faster mixing time provided by a stirred-tank
reactor. As a result stirred-tank bioreactor has become the
prevailing type used for cell culture.
Disposable Single-Use Bioreactors
Many different types of disposable single-use plastic
bioreactors including T-flasks, roller bottles, and
multiple flat panels are commonly used for production
of recombinant proteins and viral vaccines using both
attachment-dependent and suspension cells. Their surface
may be treated to facilitate attachment of adherent cell
cultures. However, for suspension cells, it is usually
more convenient to grow the cells in a stirred vessel,
and disposable plastic bioreactors are used only at
smaller scale when ease of operation is a more important
consideration.
Roller bottles are cylindrical screw-capped bottles with
a total volume ranging from 1 to 1.5 L suitable for a cul-
ture volume of 0.10.3 L. Stacks of bottles can be placed
on a rack and can be rotated at 14 rpm. For small-scale
operations, roller bottles provide many advantages for the
cultivation of adherent cells. It is relatively inexpensive
to set up. It allows for a rapid change of throughput
in response to the need. Furthermore, replacing medium
from cell growth medium to one designed for product for-
mation is rather straightforward. It is particularly useful
in the case that the serum-containing medium needs to
be replaced by a serum-free or protein-free medium for
the production of secreted proteins or viruses. The trans-
parent glass or plastic wall allows visual or microscopic
examination of the culture status. Microbial contaminated
bottles can be readily spotted and discarded before being
pooled together with others.
However, the drawbacks of roller bottles are numer-
ous for large-scale production of biologics. On-line
environmental monitoring and control is virtually
impossible, or at least impractical. The aseptic bottle
handling for inoculation protease treatment for cell
detachment and expansion, medium exchange, and
product harvest requires extensive well-trained labor to
ensure a low failure rate. Each batch of manufacturing
can easily involve hundreds or even thousands of bottles,
and the large number of manual steps involved makes
the risk of microbial contamination rather high. Despite
these significant drawbacks, roller bottles are still widely
used in the production of recombinant proteins and
viral vaccines, often because the product involved has
been approved by regulatory agencies and a process
change would incur too high a cost and may result in

product comparability issues. In some cases, roller bottles
are selected because the process is easy to implement
and the production capacity needed is manageable.
Notable examples include the production of EPO using
recombinant CHO cells and the production of the live
attenuated chickenpox (varicella) vaccine and herpes
zoster (shingles) vaccine using adherent secondary human
lung fibroblasts MRC-5 cells. Recent improvements
include developing a fully automatic and continuous
cell culture for a large number of roller bottles to make
industrial-scale production by mammalian cell culture
(29).
Nunc Cell Factories (NCFs) are widely used in lab-
oratories and in industrial production of viral vectors,
vaccines, and recombinant proteins. They are ideal to sup-
port growth of adherent cells; however, they can also be
used for suspension culture. A 40-tray NCF has a capac-
ity of 25,280 cm2 and 8000 mL liquid volume. In one
study adherent PER.C6 cells, grown on a single tray and
transfected with adenovirus plasmid, produced 5 1010
of an adenovirus type 5 vector expressing HIV-1 gag gene
(MRKAd5gag). Scale-up can be easily performed using
multiple tray systems (30). For large-scale manufacturing
using tray disposal device, a mechanical handling system
can be used. One notable example is the production of
a recently licensed multivalent Rotavirus vaccine using
Vero cells, a continuous African green monkey kidney cell
line. The vaccine is a live attenuated virus vaccine that
contains five different human-bovine virus reassortants,
each produced from a different process (31).
Another multiple plate system (CellCube Module)
entails 9 in.2 polystyrene plates stacked vertically at 1 mm
spacing in an overmolding resin case. The space between
stacks is completely filled with medium. Continuous
medium circulation is used for oxygen and nutrient
supply. It has been used in the cultivation of MRC-5
cells for the production of attenuated hepatitis A virus
(HAV) and for the production of VA2TA, an inactivated
HAV vaccine. These disposable bioreactors have also
found some popularity for the production of cells and
gene therapy vectors at intermediate scales of operation
(32,33).
Disposable cell culture wares suitable for modest
scale of suspension culture are increasingly being used
for production of biopharmaceuticals. Most of them
use disposable plastic bags as cell chambers. They are
especially suitable for cell expansion before reaching
the production scale. Wave mainly consists of a
presterilized, disposable Cellbag and a special rocking
platform. The rocking motion of this platform induces
waves in the culture fluid. These waves provide mixing
and oxygen/gas transfer to support over 20 106 cells/mL.
With no cleaning or sterilization required, dispos-
able bags simplify setup operations. Various systems
at a culture volume from 0.1 to 500 L are available
(http://www.wavebiotech.com/products/wave bioreactor/in
dex.html). pH, dissolved oxygen (DO), temperature sen-
sors are designed for these single-use bioreactors. In
manufacturing settings, validating the equipment for
restart of the process contributes to a very significant
part of overall operating cost. The use of disposable bag
MAMMALIAN CELL BIOREACTORS 7
systems can significantly shorten the process development
time, require much less capital investment than installing
stirred-tank bioreactors, and reduce the cost associated
with validation.
A more extreme case of single-use alternative to
conventional fermentor is a retrofit product to mimic
existing stainless steel bioreactor vessel. It consists of
a reusable stainless steel outer support container and
single-use BioProcess Container (BPC) with a working
volume of up to 1000 L, which can be integrated with
the existing bioreactor control system. The integrated
sparging system provides effective oxygen transfer
(http://www.hyclone.com/bpc/new prod/sub.php). Since all
parts contacting the cell culture are single-use and tested,
no cleaning or sterilization is needed.
Hollow Fiber Bioreactor
The use of hollow fiber reactors for cultivation of mam-
malian cells dates back to the early 1970s (34). A hollow
fiber system can be used for anchorage-dependent and
suspension cells. It consists of a bundle of capillary fibers
sealed inside a cylindrical tube. The basic configuration is
rather similar to the hollow fiber cartridge used in kidney
dialysis. In fact, many hollow fiber bioreactors basically
consist of a kidney dialysis hollow fiber unit connected to a
medium recirculating reservoir. The hollow fiber, in most
cases, consists of supporting polymeric porous materials
for mechanical strength and a thin layer of membrane
that provides selective passage of molecules depending on
their size. The MWCO of the membrane differs according
to applications, ranging from a few thousand to a hundred
thousand daltons. In most cases, an ultrafiltration mem-
brane is used (35). The ultrafiltration membrane prevents
free diffusion of secreted product molecules from passing
through the membrane and allows them to accumulate in
the extracapillary space to a high concentration. The cul-
ture medium is pumped usually through the fiber lumen,
and cells grow in the extracapillary space or the shell side.
Supply of low molecular weight nutrients to the cells and
the removal of waste products occur by diffusive transport
across the membrane between the lumen and the shell
spaces. Although the use of microfiltration hollow fiber
membranes for cell culture is infrequent, it does find appli-
cation in various research uses for studying metabolism
and for the cultivation of anchorage-dependent or highly
aggregated cells for which a convective flow of medium
through the extracapillary space to bathe cells in medium
is desired.
Most hollow fiber systems used for cell culture employ
fibers of approximately 250350 m in outer diameter.
The interfiber distance is at least as large. A factor lim-
iting the potential of scaling-up of a hollow fiber system
is mass transfer. In the radial direction, the nutrient
(including oxygen) diffuses through the hollow fiber wall
and membrane to reach the site of cells for consumption
by the cells. In the axial direction, the medium entrance
region is exposed to fluid with higher oxygen, while the
exit end inevitably sees a lower concentration of oxygen.
Oxygen is of primary concern because it is usually the first
nutrient to become growth limiting. To ensure ample oxy-
gen supply, the recirculating medium is typically passed
8 MAMMALIAN CELL BIOREACTORS
through an oxygenator (e.g. one used in surgery opera-
tions) before it enters the hollow fiber system. A typical
setup of a hollow fiber system therefore includes not only a
constant temperature incubator to house the hollow fiber
bioreactors but also pH control, oxygenation and oxygen
monitoring systems, and a medium reservoir for recircu-
lating medium. In addition, frequently a small medium
reservoir is used to supply high molecular weight nutrient
supplements to the extracapillary space directly. A har-
vest reservoir is used to collect the product containing fluid
from extracapillary space. In a hollow fiber bioreactor, a
nonuniform growth of cell mass and product accumulation
is often seen (36). This is usually caused by the small
convective flow in the shell side, the difficulty to inoculate
cells uniformly, and settling due to gravity.
Techniques to improve oxygen supply include the use
of fiber or silicon tubing for oxygen supply into the shell
side of the reactor. However, the use of mixed fibers in the
bioreactor makes the bioreactor harder to manufacture.
Another technique is to reverse the flow direction periodi-
cally to inverse the axial regions exposed to high and low
oxygen concentration.
Despite its limitation in scaling-up, the hollow fiber
system is a proven technology relatively easy to apply to
processes requiring a modest amount of product. In some
tissue engineering and cellular therapy applications, the
product is often patient specific and the quantity of cells
needed is relatively small. A hollow fiber system could be
ideal for those applications.
Bioreactors for Scale-Down/High Throughput Operations
Cell culture has long been carried out in small scales, such
as 96 well or even 384 well format, for various biological
assays. Such small-scale systems are ideal for parallel cul-
tures for testing medium components or other biologically
active ingredients. They are also convenient for screening
a large cell population to identify cell variants or clones
that have certain desired characteristics. Although those
small-scale systems are largely carried out as stationary
cultures like T-flask, there are also deep well systems in
such 96 or 384 well format for parallel culture of cells
kept in suspension. The use of suspension system allows
for larger quantities of cells be grown, which is important
for some cases where the assay requires a larger sample
volume. Such systems are also more amenable to opti-
mization of culture conditions. The systems conform to
the standard format of laboratory liquid handling robotic
systems and are suitable for high throughput operations
needed for drug screening, toxicity testing, or gene trans-
fection studies. These high throughput formats are also
employed in screening clones with high productivity as
well as in media development and optimization.
In the past few years, smaller scale operations are
increasingly being used in bioprocess industry as they
enable reduction in labor and material costs and expand
the number of variables that can be investigated as com-
pared to conventional laboratory bioreactors with a culture
volume varying from 0.5 to 10 L. Recent efforts on biore-
actor miniaturization also aim to reproduce the conditions
of process scale to render the studies at small scale more
relevant to manufacturing conditions.
The key parameters of design and operation in those
small-scale reactors are thus mixing, mass transfer, and
other factors affecting environmental control (such as pH,
dissolved oxygen, and nutrient levels). The most com-
monly used small-scale bioreactors that provide mixing is
the shaker flask. Mixing is carried out simply by shak-
ing the bioreactor using various shaking diameters and
angles. Although convenient for cultivating cells, conven-
tional shaker flasks require large space for a given culture
volume. The rotational speed that can be used is limited
to avoid spillage. Furthermore, the rotary motion used in
providing mixing does not provide a high oxygen trans-
fer rate. In the past decade a number of culture tube
small-scale systems became available commercially. The
simple cylindrical shape and the relatively small volume
of culture fluid allows a high shaking speed to be used.
Oxygen transfer rate can possibly be improved by using
square- versus round-shaped wells (37). A high kL a value
has been reported in square-shaped shake tubes with gas
permeable membrane for oxygen delivery (38). The volume
of such tube-based culture system ranges from a couple
to tens of milliliters. In some cases, mixing is provided by
high frequency motion in a vertical direction, instead of
the traditional rotary motion. Small tube cultures in 96
well format with mixing provide a mechanism amenable to
robot-assisted high throughput operation. However, with
a small culture volume and highly turbulent liquid mix-
ing, evaporation can be seriously affecting the osmolality
of culture. Evaporation in the plate formats is generally
prevented by using low-evaporation lids.
From an operational standpoint of view, especially ease
of sampling as well as lower risk of cross-contamination,
24 and 96 well plates are more commonly used for cell
cultivation compared to plates with more densely packed
wells (37,39). 24 MBR (manufactured by Micro-Reactor
Technologies, Inc., Mountain View, CA) and marketed by
Applikon, Inc., Foster City, CA) is an example of micro-
bioreactor system made using special 24-well micro titer
plate.
To mimic large-scale cultures, small bioreactors
employing stirred agitation using impellers driven with
a shaft or magnetic transmission are used. Stirred
minibioreactors may be constructed using wide range of
materials including glass and polymers, with a working
volume in the order of 20100 mL (40). Larger volume
compared to miniature plates allows for ease of feeding,
sampling, better instrumentation, and control. However,
relatively higher cost of the stirred minibioreactors may
limit their application for high throughput screening.
The system needs to be better optimized to provide high
quality instrumentation at a lower price.
Instead of employing ministirred bioreactors with tens
of milliliters of culture volume, some have miniaturized
mixing by employing lab-on-a-chip idea to allow for culti-
vation on polymer or glass plates (41,42). These reactors
are typically very small with 5150 L working volume
(47), and the capacity to measure critical process vari-
ables on-line and even perform chemostat cultivations
(42,43). It is worthwhile to note that the small work-
ing volumes require analytical tools capable of analyzing

correspondingly small samples. In the literature, micro-
bioreactors and larger, liter-scale cultivations comparisons
have reported equivalency for process parameters and
optical density measurements (40,44).
To be better able to manipulate the environment to
mimic large-scale operations, environmental monitoring
and control is important. Because of the small culture vol-
ume and restriction on sample volume, on-line monitoring
is highly desirable or even essential for high through-
put operations. Typically on-line monitoring of bioreactors
relies on in situ electrical probes that convert chemical
state or reaction to an electric signal. Miniaturized pH
and DO probes can be used with stirred minibioreactors
(Frachon et al., 2006). Such wired, fix-position probes are
cumbersome to use in high throughput systems employing
culture tubes or shaker flasks and are too large for 96 well
format based systems. For these microscale bioreactors,
optical sensor technology that monitors pH and dissolved
oxygen noninvasively and possibly transmits the signal
remotely is preferred. Fluorescence measurement tech-
niques for measuring dissolved oxygen and pH using dye
in culture media and/or sensing patches are being devel-
oped (42,4446). Biomass measurements can possibly be
made by using simple transmission intensity measure-
ment (42) or miniature capacitance probe. The availability
of better on-line sensing will certainly facilitate the use of
miniaturized cell culture systems.
Bioreactor Scale Translation and Intensification
As the mammalian cells become a major workhorse for
the production of biopharmaceuticals, the focus of process
research and development also shifted from the develop-
ment of bioreactors to process optimization and intensifi-
cation using existing bioreactors. In the past decade, we
have witnessed a surge in the productivity, from 100s mg/L
to 25 g/L, in the production of recombinant proteins from
cell culture processes. This increased productivity has been
the result of intensified optimization of fed-batch processes
to increase cell concentration and to extend the high pro-
ductivity stage of the culture. The advances have allowed a
much higher demand of product be met without increasing
physical manufacturing capacities and have saved large
sums of capital investment. Such fed-batch cell culture
processes are now commonly used for production of clini-
cal and commercial supplies of antibody products at scales
of up to 20,000 L. The continued advances are reliant on
process productivity enhancement and scale-down efforts.
Current protocol of generating a high producing cell line
involves isolation and evaluation of cell clones derived
from single cells after the introduction of transgene.
Clonal variation renders their behavior difficult to predict
under different culture environment. Rapid optimization
of a high productivity process and successful bioreactor
scale translation, especially predicting a particular pro-
ducing cell lines behavior in manufacturing environment,
is critical for the production of biopharmaceuticals. Cell
line characterization and process optimization are invari-
ably performed in small-scale- and pilot-scale bioreactors.
A high confidence on the translatability of small- and
pilot-scale results to manufacturing conditions will greatly
MAMMALIAN CELL BIOREACTORS 9
fasten the timelines and reduce the drug development
cost. In addition, a suitable scale-down model is needed
to support process characterization, troubleshooting, and
process validation.
A successful scale-up and scale-down depends on a
good understanding of physical and chemical parameters
affected by scale, their quantitative or qualitative rela-
tionship to scale, and the design of the scale-down model.
It is the changes in the dynamics of these critical param-
eters in scale translation that eventually cause changes
in cellular physiology and productivity. Mechanical agita-
tion and shear, aeration, and oxygen (and carbon dioxide)
transfer, which are the conventional subjects of scale-up
studies, are apparently important parameters. Addition-
ally, the parameter values for pH and gas flow control often
scale nonlinearly causing subtle differences in chemical
environment, such as dissolved carbon dioxide and osmo-
lality. There have been few reports on scale-down models,
which describe time profiles of scale-sensitive physical
and chemical parameters. It is worth noting that key to
scale-up studies is the comparison of the cellular physi-
ology in both small and large scales. The profiles of cell
growth, metabolism, and product formation have been the
key parameters being compared in scale translation. How-
ever, in the past few years we have seen various omic
tools, especially DNA microarrays, increasingly finding
their applications in bioprocessing research. Its potential
in physiological comparative studies on scale translation
is worth exploring.
CONCLUDING REMARKS
The reactors discussed above are the ones commonly used
in manufacturing of biopharmaceuticals. The selection of a
reactor for a particular process is largely based on process
needs, especially whether the cells employed are adherent
or suspension cells. Large-scale bioreactors, whether they
are used for fed-batch operations or perfusion cultures, are
mostly stirred tanks. Their dominance is attributed to the
robust performance gained through their wide use in var-
ious bioprocesses over the decades. However, many other
considerations, especially low capital investment and ease
in operations and validation, have rendered an increased
usage of disposable devices in the past few years. Many
processes of moderate scales and seed culture preparations
of large-scale culture are increasingly being performed in
disposable devices. Other types of reactors, many of which
were innovated a couple of decades ago, although not
commonly used in bioprocessing nowadays, are finding
applications in tissue engineering. Furthermore, minia-
turized bioreactors are increasingly being used in high
throughput operations. In the near future, we may see
that the experience gained in bioprocess technology begin
to benefit high throughput technology.
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