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MAMMALIAN CELL BIOREACTORS adapt to suspension growth, serum-free, even protein-free,

and other culture conditions at very large scale (over


WEICHANG ZHOU1 , GARGI 20,000 L). Over time the bioreactors used for industrial
SETH2 , MARIA J. GUARDIA3 , cell culture converge on a few basic configurations. This
and WEI-SHOU HU4 chapter focuses only on the main types used currently
1 Genzyme Corporation, for industrial cell culture. It should be noted that many
Framingham, of the bioreactors developed in the 1980s, although not
Massachusetts commonly used, might find specialized applications in
2 Genentech, Inc., 1 DNA Way, other areas, such as tissue engineering, gene therapy, and
South San Francisco, cellular therapy.
California
3 Camino De Purchill, Puleva

Biotech Department of BASIC REACTOR OPERATION AND KINETICS


Process Engineering,
Granada, Spain Basic Types of Reactors
4 University of Minnesota,

Department of Chemical Mammalian cell bioreactors can be generally categorized


Engineering and Materials similarly to chemical reactors according to their mix-
Science, Minneapolis, ing characteristics. It is instructive to review two ideal
Minnesota 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-
INTRODUCTION neous through the reactor. The mathematical description
of ideal continuous flow stirred-tank reactor is described
The year 2007 marked the 20th anniversary of the intro-
by the following first-order differential equation.
duction of the first recombinant mammalian cell culture
produced therapeutic protein, tissue plasminogen activa- d(VCA )
= Fi CAi Fo CAo + rV (1)
tor (Activase) by Genentech. The first Chinese hamster dt A

ovary (CHO) cell produced protein drug was followed by


V is the culture volume in the bioreactor, CA the concen-
the introduction of erythropoietin (EPO) (by Amgen in
tration of nutrient or product A, t is time, F is the flow
1989). In the past decade, a rapid expansion of recombi-
rate, and rA is the volumetric consumption rate of nutrient
nant antibody-based therapeutics has pushed the annual
or production rate of product A.
production amount of therapeutic protein to tens of thou-
In an ideal continuous stirred-tank reactor, there is no
sands of kilograms and over 20 billion US dollars in
flow bypass and no shunt of substrate from inlet to outlet,
revenue per annum (Tables 1 and 2). Before the advent of
no dead zones or clumps of undissolved solid substrate
recombinant DNA technology, industrial mammalian cell
floating around. Any substrate added through feeding is
culture was used primarily for the production of biologics
instantaneously distributed throughout the entire reactor,
using isolated transformed cells and viral vaccines using
and when gas sparging is employed, the agitator provides
primary cells and normal diploid cell strains. The arrival
an intimately mixed gasliquid.
of rDNA technology and the expression of heterologous
The basic model for the tubular reactor (such as hollow
proteins in mammalian cells completely transformed cell
fiber and many other membrane bioreactor systems that
culture technology. Except for the production of attenuated
are described later in this chapter) is that liquid phase
or inactivated viral vaccines, virtually all mammalian cell
moves as a plug-flow, meaning that there is neither varia-
culture processes now employ continuous cell lines, most of
tion of axial velocity over the cross section nor backmixing
which have been adapted to grow in suspension, without
a surface support. The adoption of the growth of sus- in the longitudinal direction. The mass balance for com-
pension cells as the typical vehicle for the production of ponent A in a volume element Sz that described an ideal
complex molecules requiring extensive posttranslational plug-flow reactor is as follows:
modification also transformed the concept of bioreactor CA CA
selection. = vZ S + rA (2)
t z
Most research and many commercial efforts on devel-
oping bioreactors for mammalian cell culture occurred in where vz is the linear velocity in the z direction and S is
the 1980s and early 1990s. The anticipation was that the the cross-sectional area.
potentially detrimental effect of the air sparging on mam- Given the initial condition in Equation 1, or both initial
malian cells would require a newly designed bioreactor to and boundary condition in Equation 2, one can obtain the
accommodate the manufacturing needs of the new mam- concentration profiles over time; or for tubular reactors,
malian cell-derived products. What was unanticipated to the profile can be obtained over time and spatial posi-
researchers and altered the landscape of bioprocessing tion. At steady state (i.e. cell concentration and cellular
technology was the extraordinary capability of cells to activities are not changing with time), the equation for
tubular reaction becomes

Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, edited by Michael C. Flickinger
Copyright 2010 John Wiley & Sons, Inc. 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 regation where there is no interaction between different
volume elements in the bioreactor. An ideal plug-flow reac-
which describes changes of concentration of A along the tor is assumed to be under conditions of total segregation.
direction of fluid flow. It is clear that the nutrient con- Most bioreactor systems have a mixing pattern between
centration will decrease from inlet to the distal end of the two extremes and are under partially segregated con-
the reactor, while metabolite or product concentration ditions.
increases. The length of the reactor is limited because In general, laboratory and small pilot plant bioreactors
eventually nutrient depletion or metabolite accumulation are used for process development, optimization, and char-
inhibits growth and metabolism. acterization. The fluid mixing characteristics are rather
These ideal cases of well-mixed tanks or plug-flow tubu- sensitive to the scale of the reactor. Furthermore, plug-flow
lar reactors are situations that can be approximated in bioreactors are intrinsically more difficult to scale up than
small-scale laboratory conditions. The conditions in larger mixing vessels, as the concentration gradient of essen-
scale reactors deviate significantly from these ideal condi- tial nutrients, oxygen in particular, will inevitably become
tions. limiting in the downstream region of the reactor. In con-
In a well-mixed bioreactor, there are no concentration sidering the selection of bioreactors for mammalian cell
gradients in either the gas or the liquid phase. In other cultures, the mixing characteristics and their relationship
words, none of the chemical species or cells is segregated to scale-up have to be kept in mind.
MAMMALIAN CELL BIOREACTORS 3

Batch/Fed-batch Versus Continuous/Perfusion Operations the concentrated cell stream back to the reactor, while
discarding the purged cells along with the other stream.
Mammalian cell cultures can be operated in batch,
As the scale increases the holding time in the external
fed-batch, and continuous modes. In a batch culture all
recirculating loop also becomes longer and potential oxy-
nutrients are added at the beginning of a production cycle,
gen starvation must be avoided to minimize the risk of cell
except that oxygen is supplied continuously by diffusion
progression to apoptosis. Such concern may require the
through the interface with an oxygen carrying stream,
reduction of temperature in the external loop. Using an
which is part of the recirculating medium or air flowing
internal cell retention device minimizes such a concern.
through the culture. Using a conventional medium,
However, all cell retention devices may face occasional
the typical concentrations achieved in a batch culture
problems of cell clogging or mechanical failure. An exter-
are rather low, around 2 4 106 cells/mL. In many
nal device is in general easier to be replaced or duplicated
cases, batch operation tends to be run as extended batch
for alternating operation than an internal device. When a
processes that are sometimes referred to as repeated batch
culture is operated in a continuous mode, it is desirable
cultures with intermittent harvest. In this operation mode,
for the operation to last for months; a replaceable cell
spent medium with or without cells may be harvested
retention device can make the process more robust.
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
CELL SUPPORT FOR MIXING VESSELS
reactor. With this technique it is possible to prevent the
inhibitory products from accumulating to an exceedingly
Microcarriers
high level and to provide nutrients for cell growth and
production. The use of microcarriers for cell culture was first demon-
To prolong the culture period, fed-batch operation is strated four decades ago (10). The basic concept is to
practised. The culture is started at less than full vol- allow cells to attach to the surface of small suspended
ume; after a short period of growth, concentrated nutrient beads so that conventional stirred-tank bioreactors can
stream containing amino acids, glucose, and some other be used for cell cultivation. The diameter and density of
nutrients is added continuously or intermittently until these cell-laden microcarriers are usually in the range
reaching maximum volume. The total amount of nutri- of 100300 m and 1.021.05 g/cm3 so that the settling
ents added to the reactor is typically substantially (a velocity is in the order of centimeter per minute. This
few fold) higher than if the reactor was filled with fresh diameter range also gives a good growth surface area
basal medium at the beginning. Such fed-batch processes per reactor volume. Even at a moderate microcarrier con-
reach higher cell concentrations and prolong the prod- centration, in the range of 815% culture volume being
uct formation period. The final product concentration is occupied by microcarriers, a significantly larger surface
substantially higher than that in a batch culture. area per reactor volume can be achieved in a microcarrier
Continuous chemostat is a very useful research tool. culture than in T-flasks, roller bottles, or other plastic
Once a steady state is reached, the spent medium with- reactors with flat surfaces.
drawn is balanced by equal supply rate of fresh medium, Although even smaller microcarriers, less than
and the amount of cells being continuously discharged 100 m in diameter, can provide more surface area, they
from the bioreactor is balanced by growth. A drawback are not used often. Most adherent-dependent cells do not
of a simple continuous culture is the low cell concentra- develop their normal morphology, and in many cases,
tion, and the corresponding low product throughput of the do not multiply well on surfaces with an excessively
system. To increase the cell concentration and the reactor high curvature. These cells do not grow well on small
throughput, a perfusion culture is more frequently used in microcarriers. On the other hand, some cells, especially
industrial continuous operations. In a perfusion culture, transformed cells, multiply well even though they are
a high medium flow rate (one much higher than the cell attached to small microcarriers and do not spread.
growth rate, which would wash out cells in a simple con- These cells, after attaching to the small microcarriers,
tinuous culture) is used, but a cell retention device is used agglomerate to form aggregates and continue to grow to
to prevent most of the cells from leaving the reactor. The high density. The small microcarriers, usually with a
cell concentration in the reactor is many times higher than diameter of about 50 m, serve as nuclei for the initiation
that for a simple continuous culture. of aggregate formation (11).
A cell retention device may be physically positioned The microcarriers can be made of many different mate-
inside or outside of the bioreactor vessel (1,2). The devices rials, including dextran, gelatin (12), polystyrene (13,14),
that have been used include settling tanks (3), external glass (15), and cellulose (16). Not all these are com-
centrifuges (4) (5), external membrane filtration (6), inter- mercially available. In general, in addition to having a
nal microfiltration (7), rotating wire cage (spin filters) wettable surface, the backbone materials of microcar-
(8), and acoustic settling device (9). All these, or similar riers often need to be chemically modified to improve
devices, have been shown to be effective at laboratory scale. cell attachment. One of the most widely used microcarri-
On the industrial scale, bioreactors of up to hundreds of ers, the dextran based ones, are derivatized with charged
liters, centrifuges, settling devices, and spin filters appear molecules or collagen. Polystyrene microcarriers have also
to be the top choices. The employment of a centrifuge or a been coated with collagen or other adhesion molecules for
settling device necessitates a recirculation loop to return better performance.
4 MAMMALIAN CELL BIOREACTORS

An advantage of microcarriers for the industrial-scale Aggregates


culture of anchorage-dependent cells is the ease of
Some transformed cells that normally attach to or spread
separating cells from culture medium. Many microcarrier
and grow on a surface can form aggregates when culti-
cultures require medium exchanges during cultivation
vated on less hydrophilic surfaces or in shaker flasks or
to remove accumulating lactate, ammonia, and other
stirred vessels (19). Different methods have been used to
metabolites and to replenish nutrients. In many cases,
induce aggregate formation for cells. Aggregation can be
the cell-laden microcarriers are simply allowed to settle promoted by manipulating the calcium concentration (20)
and the spent medium is withdrawn and replenished. in conjunction with the agitation rate. Aggregate cultures
In large-scale operations, continuous or semicontin- have advantages similar to microcarrier cultures. They
uous perfusion is more frequently used. This can be can be cultivated in conventional stirred-tank reactors
accomplished by withdrawing medium through a coarse with environmental control. They can be allowed to set-
screen that allows medium to pass through but retains tle relatively rapidly by stopping agitation. This permits
microcarriers in the reactor. Many types of cells have been medium replenishment readily.
grown on microcarriers, including fibroblasts, kidney, For many cell types, one limitation for application of
epithelial, hepatocyte, neuroblastoma, and endothelial the aggregate culture is that the rate of aggregate for-
cells from various species. Overall, microcarrier culture is mation is slow. One way to induce aggregate formation
a convenient laboratory and research tool, and it has the is to add microspheres, the small microcarriers with a
advantage of being amenable to large-scale production if diameter of about 50 m, to cell suspension to allow for
a large quantity of product is needed. 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
Macroporous Microcarriers
influence the viral infection kinetic and yield for vaccine
A variant of microcarriers is one with large pores of tens formation (21). Many cell lines, including BHK, CHO, 293,
of micrometers in its interior. The void space inside allows and ST cells, have been cultivated as aggregates with sizes
cells to be cultivated not only on the external surface but ranging between 90 and 400 m, without the formation of
also internally. Cells in the interior are less susceptible to necrotic centers (11,22).
mechanical damage caused by agitation and gas sparging.
Of course, being in the interior of microcarriers, cells are Cell Entrapment
more likely to be subject to oxygen limitation due to the Another method of cell cultivation, which enables the
long diffusional distance, especially since most macrop- use of a stirred-tank reactor, is cell entrapment. This
orous microcarriers have a larger diameter (500 m to technique entails entrapping cells in polymeric spheres.
a couple of millimeters). Macroporous microcarriers are The spheres can then be coated with another polymeric
made of different materials, including gelatin (17), colla- film with controlled cross-linking of the polymer to mod-
gen (18), and plastic (e.g. Cytoline). Some macroporous ulate the permeability to molecule according to their
microcarriers have apparent bulk density similar to that size. This is commonly referred to as microencapsula-
of conventional microcarriers. However, because of their tion. In other applications, the spheres with entrapped
larger particle size, the settling velocity is substantially cells are cultivated in the same way as the beads sus-
higher. In some cases, the settling velocity is orders of pended in medium to allow for cell growth. One of the
magnitude higher, in the order 1001000 cm/min. These polymers frequently used for cell entrapment is calcium
microcarriers are suitable for use in fluidized beds with alginate. Cell entrapment in calcium alginate is accom-
a high fluid recirculation rate. Because of the high set- plished by preparing a cell suspension in sodium alginate
tling velocity even at high upward supervelocity, the and adding it, in a drop-wise fashion, into a solution
carrier can be retained in the reactor. Similar to micro- of calcium chloride. A high calcium concentration pro-
carriers many cell lines have been successfully grown on motes cross-linking of alginate, instantaneously forming
macroporous microcarriers including Vero, HepG2, CHO, beads containing entrapped cells. The alginate beads may
and HEK293 cells. The final cell concentration achieved be coated with polylysine for increased mechanical char-
tends to be higher than that obtained with an equivalent acteristics and structural integrity, but such treatment
volumetric concentration of conventional microcarriers. decreases the molecular weight cutoff (MWCO) and pro-
But, in some cases, the growth kinetics are a bit slower hibits large-molecular weight proteins in the medium
because the penetration of cells into the interior may be from reaching the cells. To prepare hollow spheres, the
slowed or even retarded by restrictive opening of these alginate gel inside a bead coated with a polylysine is liq-
pores. uefied by treatment with a calcium chelator such as EDTA
Since the cells are protected inside the capsules, they (ethylenediaminetetraacetic acid) or citrate.
are protected from hydrodynamic damage. The culture The diameter of these beads is often in the order of
can be stirred at faster rates than conventional culture, millimeters. The mechanical strength of the gel which con-
which improves nutrient, metabolite, and gas exchange. stitutes the beads is relatively weak even with polymeric
Entrapped cells may be cultured in stirred-tank bioreac- coating. Large-scale application using such techniques is
tors, fixed-bed and fluidized-bed reactors. An airlift reactor not easy. On the other hand, the microcapsule provides
could be used as well, provided that the beads are small a means of immunoisolation of transplanted cells or tis-
and not significantly denser than the medium. 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 Figure 1. Schematic of a stirred-tank bioreactor. (Courtesy of
eddies may be severe. Thus, fluid shear is of paramount Genentech, Inc.)
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 culture. The use of pitched blade, as opposed to Rushton
of the microcarriers (26). The microcarriermicrocarrier turbine, reflects the different purposes of agitation. In
collisions are also expected to increase in frequency and microbial fermentation, agitation is needed at a higher
severity in the presence of eddies of size equal to the power input to disperse air bubbles and to increase oxygen
average interparticle distance. Suspended cells are gen- transfer efficiency, whereas in mammalian cell culture,
erally less prone to hydrodynamic damage than cells on a much lower agitation rate is sufficient to keep cells or
microcarriers. Overall, stirred-tank bioreactors are most microcarriers suspended in medium relatively uniformly
commonly used for cell cultivation. The use of disposable and to support the oxygen demand of the culture. In gen-
cell culture devices (also referred to as a single-use device) eral, the acceptable mixing time in a mammalian cell
suitable for process scale (up to hundreds of liters) has culture bioreactor is substantially higher than that in a
become common place in the past few years, especially in microbial fermentor. The oxygen transfer capacity in a cell
cell propagation for inoculum preparation. culture bioreactor is substantially lower than that in a
microbial fermentor, since the typical oxygen demand in a
Stirred-Tank Bioreactor mammalian cell culture is also much lower (1050 times)
Stirred tanks have been widely used for culturing sus- due to lower cell mass than that in microbial fermentation.
pension cells since the 1960s. The basic configuration of The most efficient way to supply oxygen, especially in
stirred-tank bioreactors for mammalian cell culture is large scale, is the direct sparging of air or oxygen-enriched
rather similar to that of microbial fermentors (Fig. 1). The air. Direct sparging, however, can potentially damage
aspect ratio (the height-to-diameter ratio) of bioreactors cells. Numerous studies have tried to clarify the mech-
used for mammalian cell culture is often smaller than anism of cell damage caused by direct gas sparging (27).
that for microbial cultivation. However, in recent years The high energy released as the gas bubbles release from
that distinction has become less apparent as the scale of the liquid surface due to bubble ruptures appears to be
cell culture bioreactors approaches tens of cubic meters responsible for its most damaging effects. These conse-
in volume. However, the bioreactors used for microcarrier quences can be partially alleviated by the addition of
cultures generally have a low aspect ratio to provide a polyols in the culture medium to prevent cells from attach-
higher surface-to-volume ratio. The power input per unit ing to the bubble surface. It is, in fact, standard practice
volume of bioreactor is substantially lower in mammalian to include Pluronic F-68 in low protein or protein-free
cell culture bioreactors. While the Rushton type impeller is medium for suspension cultures. This effectively prevents
the norm in microbial fermentors, mammalian cell culture cells from attaching to the bubble surface, thus minimizing
bioreactors mostly employ pitched blade or marine type cell damage or death due to bubble ruptures. Commonly
impeller. Like in microbial fermentors, multiple impellers used spargers include open pipe, ring sparger with multi-
are used in larger scale bioreactors for mammalian cell ple holes of a diameter of 1/16 in., and microsparger made
6 MAMMALIAN CELL BIOREACTORS

of sintered stainless steel. Microspargers create swarms of BHK 21, human lymphoblastoid, CHO, hybridomas,
of small bubbles and are efficient for oxygen transfer, as and insect cells. In the past decade, the cell concentration
they create a larger interfacial area at a given air flow achieved in cell culture bioreactor increased substantially
rate. With the use of microspargers, the gas flow rate as fed-batch culture became the prevailing mode of opera-
required to supply oxygen to maintain certain dissolved tion. In a fed-batch mode, the culture is often initiated at
oxygen levels tends to be smaller, leading to a lower CO2 about two thirds of the maximum volume. The other one
removal rate and a generally higher CO2 concentration in third is gradually fed throughout the cultivation period.
culture. Furthermore, in larger vessels, bubble coalescence The initially low volume makes the use of airlift biore-
is unavoidable, thus offsetting the advantage of small bub- actors impractical as the draft tube is not completely
bles. The open pipe or multiple-hole spargers are simple submerged in the medium. Furthermore, the increased
and are generally used in large reactors. In general, gas oxygen consumption caused by the high cell concentration
flow rates for sparging can be scaled up by keeping the requires a faster mixing time provided by a stirred-tank
same superficial gas velocity. However, it is important to reactor. As a result stirred-tank bioreactor has become the
increase the number of holes for sparging linearly to the prevailing type used for cell culture.
gas flow rates in order to keep the gas entrance velocity
at the same levels as it has been reported recently that a Disposable Single-Use Bioreactors
high gas entrance velocity of more than 30 m/s may exert
an adverse effect on NS0 cells (28). Many different types of disposable single-use plastic
bioreactors including T-flasks, roller bottles, and
Airlift Bioreactor multiple flat panels are commonly used for production
of recombinant proteins and viral vaccines using both
An airlift reactor is essentially a relatively high aspect attachment-dependent and suspension cells. Their surface
ratio (tall and thin) tank with an internal concentric draft may be treated to facilitate attachment of adherent cell
tube as a guide for fluid recirculation (Fig. 2). The internal cultures. However, for suspension cells, it is usually
liquid circulation is achieved by sparging at the bottom of more convenient to grow the cells in a stirred vessel,
the draft tube. The sparged section has a lower effective and disposable plastic bioreactors are used only at
density than the bubble-free section, and the difference smaller scale when ease of operation is a more important
in hydrostatic pressure between the two sections induces consideration.
the liquid circulation upward in the sparged section (riser) Roller bottles are cylindrical screw-capped bottles with
and downward in the bubble-free section (downcomer). The a total volume ranging from 1 to 1.5 L suitable for a cul-
loop has the advantages of permitting high efficiency mass ture volume of 0.10.3 L. Stacks of bottles can be placed
transfer and improving the flow and mixing properties in on a rack and can be rotated at 14 rpm. For small-scale
the vessel. A potential advantage of airlift reactors is the operations, roller bottles provide many advantages for the
low capital costs because of their simple mechanical con- cultivation of adherent cells. It is relatively inexpensive
figuration. Although simple in construction, sound design to set up. It allows for a rapid change of throughput
is critical for optimal hydrodynamic behavior. Airlift reac- in response to the need. Furthermore, replacing medium
tors have been used successfully with suspension cultures 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
Air viral vaccines, often because the product involved has
been approved by regulatory agencies and a process
Figure 2. Schematic of an airlift bioreactor. change would incur too high a cost and may result in
MAMMALIAN CELL BIOREACTORS 7

product comparability issues. In some cases, roller bottles systems can significantly shorten the process development
are selected because the process is easy to implement time, require much less capital investment than installing
and the production capacity needed is manageable. stirred-tank bioreactors, and reduce the cost associated
Notable examples include the production of EPO using with validation.
recombinant CHO cells and the production of the live A more extreme case of single-use alternative to
attenuated chickenpox (varicella) vaccine and herpes conventional fermentor is a retrofit product to mimic
zoster (shingles) vaccine using adherent secondary human existing stainless steel bioreactor vessel. It consists of
lung fibroblasts MRC-5 cells. Recent improvements a reusable stainless steel outer support container and
include developing a fully automatic and continuous single-use BioProcess Container (BPC) with a working
cell culture for a large number of roller bottles to make volume of up to 1000 L, which can be integrated with
industrial-scale production by mammalian cell culture the existing bioreactor control system. The integrated
(29). sparging system provides effective oxygen transfer
Nunc Cell Factories (NCFs) are widely used in lab- (http://www.hyclone.com/bpc/new prod/sub.php). Since all
oratories and in industrial production of viral vectors, parts contacting the cell culture are single-use and tested,
vaccines, and recombinant proteins. They are ideal to sup- no cleaning or sterilization is needed.
port growth of adherent cells; however, they can also be
used for suspension culture. A 40-tray NCF has a capac- Hollow Fiber Bioreactor
ity of 25,280 cm2 and 8000 mL liquid volume. In one The use of hollow fiber reactors for cultivation of mam-
study adherent PER.C6 cells, grown on a single tray and malian cells dates back to the early 1970s (34). A hollow
transfected with adenovirus plasmid, produced 5 1010 fiber system can be used for anchorage-dependent and
of an adenovirus type 5 vector expressing HIV-1 gag gene suspension cells. It consists of a bundle of capillary fibers
(MRKAd5gag). Scale-up can be easily performed using sealed inside a cylindrical tube. The basic configuration is
multiple tray systems (30). For large-scale manufacturing rather similar to the hollow fiber cartridge used in kidney
using tray disposal device, a mechanical handling system dialysis. In fact, many hollow fiber bioreactors basically
can be used. One notable example is the production of consist of a kidney dialysis hollow fiber unit connected to a
a recently licensed multivalent Rotavirus vaccine using medium recirculating reservoir. The hollow fiber, in most
Vero cells, a continuous African green monkey kidney cell cases, consists of supporting polymeric porous materials
line. The vaccine is a live attenuated virus vaccine that for mechanical strength and a thin layer of membrane
contains five different human-bovine virus reassortants, that provides selective passage of molecules depending on
each produced from a different process (31). their size. The MWCO of the membrane differs according
Another multiple plate system (CellCube Module) to applications, ranging from a few thousand to a hundred
entails 9 in.2 polystyrene plates stacked vertically at 1 mm thousand daltons. In most cases, an ultrafiltration mem-
spacing in an overmolding resin case. The space between brane is used (35). The ultrafiltration membrane prevents
stacks is completely filled with medium. Continuous free diffusion of secreted product molecules from passing
medium circulation is used for oxygen and nutrient through the membrane and allows them to accumulate in
supply. It has been used in the cultivation of MRC-5 the extracapillary space to a high concentration. The cul-
cells for the production of attenuated hepatitis A virus ture medium is pumped usually through the fiber lumen,
(HAV) and for the production of VA2TA, an inactivated and cells grow in the extracapillary space or the shell side.
HAV vaccine. These disposable bioreactors have also Supply of low molecular weight nutrients to the cells and
found some popularity for the production of cells and the removal of waste products occur by diffusive transport
gene therapy vectors at intermediate scales of operation across the membrane between the lumen and the shell
(32,33). spaces. Although the use of microfiltration hollow fiber
Disposable cell culture wares suitable for modest membranes for cell culture is infrequent, it does find appli-
scale of suspension culture are increasingly being used cation in various research uses for studying metabolism
for production of biopharmaceuticals. Most of them and for the cultivation of anchorage-dependent or highly
use disposable plastic bags as cell chambers. They are aggregated cells for which a convective flow of medium
especially suitable for cell expansion before reaching through the extracapillary space to bathe cells in medium
the production scale. Wave mainly consists of a is desired.
presterilized, disposable Cellbag and a special rocking Most hollow fiber systems used for cell culture employ
platform. The rocking motion of this platform induces fibers of approximately 250350 m in outer diameter.
waves in the culture fluid. These waves provide mixing The interfiber distance is at least as large. A factor lim-
and oxygen/gas transfer to support over 20 106 cells/mL. iting the potential of scaling-up of a hollow fiber system
With no cleaning or sterilization required, dispos- is mass transfer. In the radial direction, the nutrient
able bags simplify setup operations. Various systems (including oxygen) diffuses through the hollow fiber wall
at a culture volume from 0.1 to 500 L are available and membrane to reach the site of cells for consumption
(http://www.wavebiotech.com/products/wave bioreactor/in by the cells. In the axial direction, the medium entrance
dex.html). pH, dissolved oxygen (DO), temperature sen- region is exposed to fluid with higher oxygen, while the
sors are designed for these single-use bioreactors. In exit end inevitably sees a lower concentration of oxygen.
manufacturing settings, validating the equipment for Oxygen is of primary concern because it is usually the first
restart of the process contributes to a very significant nutrient to become growth limiting. To ensure ample oxy-
part of overall operating cost. The use of disposable bag gen supply, the recirculating medium is typically passed
8 MAMMALIAN CELL BIOREACTORS

through an oxygenator (e.g. one used in surgery opera- The key parameters of design and operation in those
tions) before it enters the hollow fiber system. A typical small-scale reactors are thus mixing, mass transfer, and
setup of a hollow fiber system therefore includes not only a other factors affecting environmental control (such as pH,
constant temperature incubator to house the hollow fiber dissolved oxygen, and nutrient levels). The most com-
bioreactors but also pH control, oxygenation and oxygen monly used small-scale bioreactors that provide mixing is
monitoring systems, and a medium reservoir for recircu- the shaker flask. Mixing is carried out simply by shak-
lating medium. In addition, frequently a small medium ing the bioreactor using various shaking diameters and
reservoir is used to supply high molecular weight nutrient angles. Although convenient for cultivating cells, conven-
supplements to the extracapillary space directly. A har- tional shaker flasks require large space for a given culture
vest reservoir is used to collect the product containing fluid volume. The rotational speed that can be used is limited
from extracapillary space. In a hollow fiber bioreactor, a to avoid spillage. Furthermore, the rotary motion used in
nonuniform growth of cell mass and product accumulation providing mixing does not provide a high oxygen trans-
is often seen (36). This is usually caused by the small fer rate. In the past decade a number of culture tube
convective flow in the shell side, the difficulty to inoculate small-scale systems became available commercially. The
cells uniformly, and settling due to gravity. simple cylindrical shape and the relatively small volume
Techniques to improve oxygen supply include the use of culture fluid allows a high shaking speed to be used.
of fiber or silicon tubing for oxygen supply into the shell Oxygen transfer rate can possibly be improved by using
side of the reactor. However, the use of mixed fibers in the square- versus round-shaped wells (37). A high kL a value
bioreactor makes the bioreactor harder to manufacture. has been reported in square-shaped shake tubes with gas
Another technique is to reverse the flow direction periodi- permeable membrane for oxygen delivery (38). The volume
cally to inverse the axial regions exposed to high and low
of such tube-based culture system ranges from a couple
oxygen concentration.
to tens of milliliters. In some cases, mixing is provided by
Despite its limitation in scaling-up, the hollow fiber
high frequency motion in a vertical direction, instead of
system is a proven technology relatively easy to apply to
the traditional rotary motion. Small tube cultures in 96
processes requiring a modest amount of product. In some
well format with mixing provide a mechanism amenable to
tissue engineering and cellular therapy applications, the
robot-assisted high throughput operation. However, with
product is often patient specific and the quantity of cells
a small culture volume and highly turbulent liquid mix-
needed is relatively small. A hollow fiber system could be
ing, evaporation can be seriously affecting the osmolality
ideal for those applications.
of culture. Evaporation in the plate formats is generally
Bioreactors for Scale-Down/High Throughput Operations prevented by using low-evaporation lids.
From an operational standpoint of view, especially ease
Cell culture has long been carried out in small scales, such of sampling as well as lower risk of cross-contamination,
as 96 well or even 384 well format, for various biological
24 and 96 well plates are more commonly used for cell
assays. Such small-scale systems are ideal for parallel cul-
cultivation compared to plates with more densely packed
tures for testing medium components or other biologically
wells (37,39). 24 MBR (manufactured by Micro-Reactor
active ingredients. They are also convenient for screening
Technologies, Inc., Mountain View, CA) and marketed by
a large cell population to identify cell variants or clones
Applikon, Inc., Foster City, CA) is an example of micro-
that have certain desired characteristics. Although those
bioreactor system made using special 24-well micro titer
small-scale systems are largely carried out as stationary
plate.
cultures like T-flask, there are also deep well systems in
To mimic large-scale cultures, small bioreactors
such 96 or 384 well format for parallel culture of cells
kept in suspension. The use of suspension system allows employing stirred agitation using impellers driven with
for larger quantities of cells be grown, which is important a shaft or magnetic transmission are used. Stirred
for some cases where the assay requires a larger sample minibioreactors may be constructed using wide range of
volume. Such systems are also more amenable to opti- materials including glass and polymers, with a working
mization of culture conditions. The systems conform to volume in the order of 20100 mL (40). Larger volume
the standard format of laboratory liquid handling robotic compared to miniature plates allows for ease of feeding,
systems and are suitable for high throughput operations sampling, better instrumentation, and control. However,
needed for drug screening, toxicity testing, or gene trans- relatively higher cost of the stirred minibioreactors may
fection studies. These high throughput formats are also limit their application for high throughput screening.
employed in screening clones with high productivity as The system needs to be better optimized to provide high
well as in media development and optimization. quality instrumentation at a lower price.
In the past few years, smaller scale operations are Instead of employing ministirred bioreactors with tens
increasingly being used in bioprocess industry as they of milliliters of culture volume, some have miniaturized
enable reduction in labor and material costs and expand mixing by employing lab-on-a-chip idea to allow for culti-
the number of variables that can be investigated as com- vation on polymer or glass plates (41,42). These reactors
pared to conventional laboratory bioreactors with a culture are typically very small with 5150 L working volume
volume varying from 0.5 to 10 L. Recent efforts on biore- (47), and the capacity to measure critical process vari-
actor miniaturization also aim to reproduce the conditions ables on-line and even perform chemostat cultivations
of process scale to render the studies at small scale more (42,43). It is worthwhile to note that the small work-
relevant to manufacturing conditions. ing volumes require analytical tools capable of analyzing
MAMMALIAN CELL BIOREACTORS 9

correspondingly small samples. In the literature, micro- fasten the timelines and reduce the drug development
bioreactors and larger, liter-scale cultivations comparisons cost. In addition, a suitable scale-down model is needed
have reported equivalency for process parameters and to support process characterization, troubleshooting, and
optical density measurements (40,44). process validation.
To be better able to manipulate the environment to A successful scale-up and scale-down depends on a
mimic large-scale operations, environmental monitoring good understanding of physical and chemical parameters
and control is important. Because of the small culture vol- affected by scale, their quantitative or qualitative rela-
ume and restriction on sample volume, on-line monitoring tionship to scale, and the design of the scale-down model.
is highly desirable or even essential for high through- It is the changes in the dynamics of these critical param-
put operations. Typically on-line monitoring of bioreactors eters in scale translation that eventually cause changes
relies on in situ electrical probes that convert chemical in cellular physiology and productivity. Mechanical agita-
state or reaction to an electric signal. Miniaturized pH tion and shear, aeration, and oxygen (and carbon dioxide)
and DO probes can be used with stirred minibioreactors transfer, which are the conventional subjects of scale-up
(Frachon et al., 2006). Such wired, fix-position probes are studies, are apparently important parameters. Addition-
cumbersome to use in high throughput systems employing ally, the parameter values for pH and gas flow control often
culture tubes or shaker flasks and are too large for 96 well scale nonlinearly causing subtle differences in chemical
format based systems. For these microscale bioreactors, environment, such as dissolved carbon dioxide and osmo-
optical sensor technology that monitors pH and dissolved lality. There have been few reports on scale-down models,
oxygen noninvasively and possibly transmits the signal which describe time profiles of scale-sensitive physical
remotely is preferred. Fluorescence measurement tech- and chemical parameters. It is worth noting that key to
niques for measuring dissolved oxygen and pH using dye scale-up studies is the comparison of the cellular physi-
in culture media and/or sensing patches are being devel- ology in both small and large scales. The profiles of cell
oped (42,4446). Biomass measurements can possibly be growth, metabolism, and product formation have been the
made by using simple transmission intensity measure- key parameters being compared in scale translation. How-
ment (42) or miniature capacitance probe. The availability ever, in the past few years we have seen various omic
of better on-line sensing will certainly facilitate the use of tools, especially DNA microarrays, increasingly finding
miniaturized cell culture systems. their applications in bioprocessing research. Its potential
in physiological comparative studies on scale translation
is worth exploring.
Bioreactor Scale Translation and Intensification
As the mammalian cells become a major workhorse for
CONCLUDING REMARKS
the production of biopharmaceuticals, the focus of process
research and development also shifted from the develop-
The reactors discussed above are the ones commonly used
ment of bioreactors to process optimization and intensifi-
in manufacturing of biopharmaceuticals. The selection of a
cation using existing bioreactors. In the past decade, we
reactor for a particular process is largely based on process
have witnessed a surge in the productivity, from 100s mg/L
needs, especially whether the cells employed are adherent
to 25 g/L, in the production of recombinant proteins from
or suspension cells. Large-scale bioreactors, whether they
cell culture processes. This increased productivity has been
are used for fed-batch operations or perfusion cultures, are
the result of intensified optimization of fed-batch processes
mostly stirred tanks. Their dominance is attributed to the
to increase cell concentration and to extend the high pro-
robust performance gained through their wide use in var-
ductivity stage of the culture. The advances have allowed a
ious bioprocesses over the decades. However, many other
much higher demand of product be met without increasing
considerations, especially low capital investment and ease
physical manufacturing capacities and have saved large
in operations and validation, have rendered an increased
sums of capital investment. Such fed-batch cell culture
usage of disposable devices in the past few years. Many
processes are now commonly used for production of clini-
processes of moderate scales and seed culture preparations
cal and commercial supplies of antibody products at scales
of large-scale culture are increasingly being performed in
of up to 20,000 L. The continued advances are reliant on
disposable devices. Other types of reactors, many of which
process productivity enhancement and scale-down efforts.
were innovated a couple of decades ago, although not
Current protocol of generating a high producing cell line
commonly used in bioprocessing nowadays, are finding
involves isolation and evaluation of cell clones derived
applications in tissue engineering. Furthermore, minia-
from single cells after the introduction of transgene. turized bioreactors are increasingly being used in high
Clonal variation renders their behavior difficult to predict throughput operations. In the near future, we may see
under different culture environment. Rapid optimization that the experience gained in bioprocess technology begin
of a high productivity process and successful bioreactor to benefit high throughput technology.
scale translation, especially predicting a particular pro-
ducing cell lines behavior in manufacturing environment,
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