Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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properties. Therefore, it is necessary, in each production lot, to employ the

same cell line and guarantee that cell characteristics do not change between

lots. Also, in research, it is important to use deﬁned cell lines and to ensure

that they do not change by mutations over time that could alter their

properties.

To guarantee the availability of cells and the maintenance of their

characteristics, a sufﬁciently large quantity of cells is kept in master and

working cell banks, as discussed in Chapters 13 and 14. The number of

cryotubes containing cells in a working cell bank is calculated to ensure

that, for the whole expected lifespan of a product, each production lot will

be obtained starting with cells from identical cryotubes. The most widely

employed method for the conservation of cell banks is cryoconservation in

liquid nitrogen at –1968C, which avoids mutations that could potentially

alter cell characteristics.

Each production lot starts by thawing cells from a cryotube, which

usually contains 10 to 20 million cells suspended in a volume of 1–10 mL.

Thawing cells is a stage of the process that should be carried out with great

care, since it can generate a high level of cell death if performed inade-

quately.

After eliminating the cryoprotector (generally DMSO – dimethyl sulf-

oxide), cells are suspended in fresh medium at a concentration between 0.1

and 0.4 million cells per-mL, depending on the cell line and the culture

medium, to be used subsequently for incubation and propagation. This

range of inoculum concentration is used as a standard for the inoculation

of animal cell cultures, since lower concentrations result in long lag phases

and in a delay in cell growth, which is detrimental to process time and

productivity.

In general, the subsequent stages occur in small bioreactors, known

simply as ﬂasks or bottles, since they have no monitoring and control

system and are usually disposable. Apart from ﬂasks and bottles, multiwell

plates can be used to culture cells, but their use is restricted mainly to

cloning and selection of cells.

In the ﬁrst step after thawing, cells are generally propagated in station-

ary ﬂasks with approximately 10 ml working volume, with a monolayer of

cells being formed on the lower surface of the ﬂask after 2–3 days. The

cells are later transferred to larger ﬂasks, maintaining the cell concentra-

tion range mentioned above. Alternatively, cells able to grow in suspen-

sion can be thawed directly from the cryotube to small stirred ﬂasks, such

as magnetically stirred ﬂasks known as spinners.

In stationary ﬂasks, oxygen transfer to the liquid medium is limited by

the small gas–liquid interface area and by the lack of agitation. For this

reason, other types of culture systems need to be used when working

with more than 100 million cells. The type of culture system to be used

depends on the cell line characteristics, mainly if it is adherent or if it is

able to grow in suspension (Figure 9.1). For cells growing in suspension,

spinner ﬂasks are largely used, although shaker ﬂasks may also be

employed. Anchorage-dependent cells, on the other hand, are frequently

cultured in roller bottles. For the transfer of adherent cells from one ﬂask

to another, cells can be released mechanically or enzymatically, e.g. using

trypsin.

222 Animal Cell Technology

All the procedures carried out on a small scale are labor-intensive and

require skilled operators. Manipulation of the cultures is carried out in

laminar-ﬂow cabinets, to maintain asepsis. During this stage of cell

propagation/inoculum development, activities are carried out by direct

operator manipulation, so that the process is considered to be ‘‘open’’.

Thus, as a general rule, these activities occur in areas separated from those

where the intermediate and production bioreactors are installed.

Figures 9.2 and 9.3 show schemes that illustrate inoculum development

from the cryotubes to production scale for suspension and adherent cells,

respectively. In these hypothetical process schemes, the expression ‘‘pro-

duction bioreactor’’ is used arbitrarily for any of the types of bioreactor

presented in the next section of this chapter. In general, different ﬂasks

and several intermediate bioreactors are used for cell propagation to reach

the quantity of cells necessary to inoculate the production bioreactor. The

number of propagation steps is a function of the ﬁnal scale of the

production bioreactor.

Roller bottle

Area: 500–1750 cm

X 2.10 cells/cm

⫽

Stationary flask

(T-flask)

Area: 25–225 cm

X 10 cells/cm

⫽

Shaker flask

Volume: 0.05–0.5 L

X 2.10 cells/mL

⫽

Spinner flask

Volume: 0.05–2 L

X 2.10 cells/mL

⫽

Figure 9.1

Low volume bioreactors. Alt hough it is mentioned in literature that stirred ﬂasks can be used up to

20 L, they are mostly used for lower volumes (up to 2 L). Xf: typical ﬁnal cell concentration.

Cryotube

~10 cells

Expansion in

stationary flasks

~10 cells

Propagation in

stirred flasks

~2.10 cells

Production

bioreactor

Inoculum bioreactor

~10 cells

Figure 9.2

Hypothetical example of the propagation of suspension cells, using stirred-tank bioreactors in the

intermediate and ﬁnal stages. The amount of cells usually obtained at the end of each step is also

indicated.

Bioreac tors for animal cells 223

9.3 Types of bioreactors

In the last few decades, a wide variety of bioreactors have been developed,

with some of them being an attempt to mimic animal organs, and others

being an adaptation of the principles of submerged microbial fermentation

to the culture of animal cells.

The bioreactors used for animal cell cultivation can be classiﬁed accord-

ing to different criteria. One of the most useful refers to the homogeneity

of the system. All culture systems are multiphase systems, since at least

two phases are involved: a solid one (cells) and a liquid one (culture

medium). Eventually, a third phase (gas) may be present due to bubble

aeration. Bioreactors in which cells are uniformly suspended in the liquid

phase are designated homogeneous bioreactors. On the other hand, those

bioreactors in which cells are not homogeneously distributed in the entire

volume are called heterogeneous bioreactors. Table 9.1 lists different types

of bioreactors classiﬁed according to the homogeneity of the system.

Given the potential risk of contamination of a biopharmaceutical

product with residues of parts of a bioreactor in contact with the culture,

the materials used in the construction of bioreactors must respect the rules

dictated by the regulatory agencies of different countries. The manufactur-

ing process of the components of a bioreactor must be well documented

and the materials must be certiﬁed.

In the case of stainless steel, the surfaces that come in contact with the

product must be made of pharmaceutical-grade stainless steel (AISI 316L).

The average surface rugosity (Ra), deﬁned as the average distance of the

base of a surface up to the rugosity proﬁle (or the average height of bumps

on a surface), must be lower than 0.8 m. Thus, different treatments are

employed for polishing the surfaces, with the ﬁnal aim of facilitating

equipment cleaning and maintaining process asepsis. Among the treat-

ments commonly employed, electropolishing is recommended for ﬁnal

surface manipulation. This treatment does not alter macrorugosity of a

surface, but it smooths its proﬁle and adds a dense inert layer of chromium

oxide, which protects the metal from corrosion. Furthermore, electropol-

ishing eliminates hydrogen from the polished surface, decreasing the

Roller bottles

~10 –10 cells

Stationary flask

~10 –10 cells

Cryotube

~10 cells

Production bioreactor

~10 cells

Figure 9.3

Hypothetical example of the propagation of adherent cells, u sing ho llow-ﬁber modules as the

production bioreactor in the ﬁnal stage. The amount of cells usually obtained at the end of each step

is also indicated.

224 Animal Cell Technology

ability of microorganisms to grow on it. Figure 9.4 (see color section)

shows a polished surface inside an animal cell stirred-tank bioreactor.

Seals, o-rings, and valve membranes must be manufactured out of

polymers that are resistant to thermal sterilization and to the bases and

acids used in the biopharmaceutical industry. When in contact with the

product, the elastomers employed must additionally present the following

safety characteristics: the rates of release of polymer components must be

low and remain below well-established threshold levels; the components

released must be innocuous to human health, according to rules previously

established.

Another material that is frequently employed is glass. It is used in small-

scale vessels and also in sight glasses of large-scale bioreactors and tanks.

A detailed review of the materials used in the manufacturing of bioreac-

tors, as well as the surface treatments used, can be found in the literature

(Krahe, 2003).

9.3.1 Homogeneous bioreactors

Stirred-tank, air-lift, and wave bioreactors are the most important types of

homogeneous bioreactors used in animal cell cultivation, as previously

outlined in Table 9.1. One of the most important advantages of using

homogeneous bioreactors is the possibility of monitoring and controlling

the physicochemical environment to which the cells are submitted in a

much more reliable way than in heterogeneous bioreactors. Thus, it is

possible to indirectly control the physiological state of cells.

Stirred-tank bioreactors

Stirred-tank bioreactors are widely used in the modern biotechnological

industry. Most products produced from animal cells on a large scale

worldwide are manufactured in this type of bioreactor. In general, these

reactors are very similar to fermenters used in industrial submerged

culture of microorganisms, and are simple to design, having the shape of a

tank and impellers to promote mixture of the contents.

Stirred-tank bioreactors can be operated according to any of the opera-

tion modes that will be discussed later in this chapter. Figure 9.5A (see

color section) shows the typical design of a stirred-tank bioreactor, ﬁtted

with a continuous supply of gases for oxygenation and pH control and

with a gas exhaust outlet in the upper part of the equipment. In most cases,

oxygenation occurs by bubbling gases below the impeller, as will be

Table 9.1 Classiﬁcation of bioreactors according to system homogeneity

Homogeneous bioreactors Heterogeneous bioreactors

Stirred-tank bioreactors Microcarrier-based systems

Air-lift bioreactors Packed-bed bioreactors

Wave bioreactors Fluidized-bed bioreactors

Hollow-ﬁber bioreactors

Bioreactors providing surfaces for attached cell

growth (roller bottles, CellCube

, Cell Factory)

Bioreac tors for animal cells 225

discussed later. The complexity of accessories and systems in this type of

equipment is much greater than that in the schematic representation of

Figure 9.5A. Figure 9.5B (see color section) is a photograph of a 300 L

stirred-tank bioreactor.

Currently, stirred-tank bioreactors have been scaled-up to working

volumes of 20 000 L (Birch and Racher, 2006). Scale-up criteria that take

into consideration the negative effects of aeration are generally adopted

due to the large inﬂuence of bubbling and shear stress on cells. This issue

will be discussed in more detail in Section 9.5. Due to the high sensitivity

of cells, a limited number of cell lines, such as CHO and NS0, nowadays

dominate the industrial animal cell technology arena, due to their greater

robustness and shear resistance (Castilho and Medronho, 2002; Castillo

et al., 2005).

Ensuring aseptic operation is one of the relevant aspects in the operation

of animal cell bioreactors. This is a particularly difﬁcult task in large-scale

stirred-tank bioreactors where large volumes of ﬂuids are manipulated and

moving parts exist. Guaranteeing containment and minimizing the risk of

microbial contamination requires careful design of the equipment, a

rigorous compliance with strict operational procedures, and the use of

simple devices that ensure reliability in the manipulation of large volumes

of culture medium.

For the aseptic transport of ﬂuids, arrangements of diaphragm valves

especially designed for use in bioreactors and ﬁtted with steam supply for

in situ sterilization of the whole system (SIP – sterilization in place) are

used. Adequate sterilization is guaranteed by online temperature monitor-

ing and the use of pulsing valves with oriﬁces for condensate elimination,

among others. The valves are also interconnected with the lines of ﬂuids

used for cleaning in place (CIP). Safe mechanical seals or, more recently,

magnetic drives, are of great importance to guarantee system containment

in spite of the moving parts.

Other elements especially designed to ensure a high efﬁciency and

operational safety are devices that use bellow valves and diaphragm pumps

that do not have primary moving parts in contact with the culture

medium. More details regarding these and other devices are available in

the technical literature (Krahe, 2003).

Validation of systems is obligatory in the production of biopharmaceu-

ticals and, thus, the bioreactors must be ‘‘validatable.’’ In the case of

stirred-tank bioreactors, apart from the functional validation, the cleaning

and sterilization procedures have to be validated, since these are repeated

use equipment. These requirements pose additional challenges to the

process engineers.

Wave bioreactors

This type of bioreactor consists of two main parts: a disposable sterile bag

made of ﬂexible plastic, in which cells are cultivated, and a moving

platform that supports and shakes the plastic bag. A hydrophobic ﬁlter

attached to the bag allows the supply of gases, and small tubing allows

culture sampling, as well as nutrient addition and product harvesting.

226 Animal Cell Technology

The plastic bag is disposable and is provided sterile by the manufacturer.

This makes it a very attractive bioreactor type for production purposes,

especially if the production scale is not very large. Since it is not sterilized

in-house, there is no need for validation of the sterilization procedure. The

validation of bag sterilization is the responsibility of its manufacturer.

Since it is not reused, there is no need for cleaning and validation of

cleaning procedures. Figure 9.6 (see color section) shows a photograph and

a schematic representation of a wave bioreactor.

The oscillatory movement of the platform generates waves in the gas–

liquid interface inside the plastic bag. This type of agitation avoids dead

zones, maintains cells in suspension and ensures adequate homogeneity,

under low shear conditions. Furthermore, the platform can control the

culture temperature. The area of the gas–liquid interface, in view of the

waves generated by agitation, is several times larger than that of the surface

when stationary. This, together with the movement caused in the cell

suspension near to the gas interface, signiﬁcantly increases oxygen trans-

fer. Volumetric oxygen transfer coefﬁcients (K

a) in the range of 3–4 h

–1

were measured by Singh (1999) for bags with working volumes in the

range of 1–100 L, suggesting that cell concentrations up to 7 3 10

cell

–1

could be obtained in this type of bioreactor under nonlimiting

conditions with respect to oxygen supply.

However, scale-up of this type of system is limited due to the fact that

oxygen transfer is a function of the area of the wave-containing liquid

surface. Since the area increases with the square and the volume with the

third power of a linear dimension, it is expected that this technology will

reach a scale limit. Nevertheless, the companies that market this type of

bioreactor offer bioreactors up to at least 500 L working volume (Wave

Biotech, 2006).

Recently, approaches to operate wave bioreactors in perfusion mode

have been proposed. This turns this type of bioreactor into an attractive

variant when there is a need for rapidly establishing a process, on a

relatively small scale, and there are no robust facilities for the supply of

auxiliary services.

The manipulation of this bioreactor is generally performed in a laminar-

ﬂow cabinet, and is thus an open and risky operation. This is probably a

characteristic that differentiates this bioreactor type from the other homo-

geneous bioreactors, where reliability of all seals and connections can be

ensured through the use of direct steam. Also, the large number of

connections using ﬂexible tubing makes the operation of this type of

bioreactor more complicated than that of other bioreactors of the same

class. Currently, the performance and application potential of these

bioreactors are under evaluation by the biotechnological community.

Air-lift bioreactors

These bioreactors are characterized by their large height-to-diameter ratio

and by an internal concentric cylinder, as shown in Figure 9.7. In the lower

central region, there is a tube that injects gas inside the bioreactor. The

pressurized gas stream generates bubbles that result in a low-density

region inside the central tube. When these bubbles ascend, the liquid is

Bioreac tors for animal cells 227

pushed upwards through the internal cylinder and returns downwards

through the external annulus, generating a characteristic ﬂow pattern, as

shown in Figure 9.7. Culture oxygenation occurs effectively due to the

contact of the cell suspension with ascending bubbles. Furthermore,

homogenization of the cell suspension is not due to mechanical agitation,

but simply due to the bioreactor geometry combined with the gas stream

injected in its bottom region.

These bioreactors are characterized by low levels of cell damage and can

also be operated in perfusion mode. However, the main limitation of this

type of equipment is related to its scale-up: air-lift bioreactors up to only

5000 L have been described so far.

Air-lift bioreactors were popular in the 1990s, but currently their large-

scale use is limited. Only the company Lonza (originally Celltech) uses

this type of bioreactor on a large scale, for the production of monoclonal

antibodies (mAbs) and other therapeutic proteins (Lonza, 2006).

9.3.2 Heterogeneous bioreactors

In this type of bioreactor, there is a compartment where cells remain

attached to a surface or immobilized on or inside a biocompatible bed.

Culture medium has to be pumped through this compartment for cells to

have access to nutrients and dissolved oxygen. The main disadvantages of

these bioreactors, developed for the cultivation of adherent cells that

require a surface for proliferation, are the impossibility of obtaining homo-

geneous samples of the cell population and the limitations of scale-up.

Gas inlet

Exhaust gas

outlet

Figure 9.7

Scheme of an air-lift bioreactor.

228 Animal Cell Technology

Microcarriers

Microcarriers are small particles, made of materials such as cellulose,

dextran, glass, collagen, or gelatin. Generally, they have a spherical shape

and present a surface structure and composition that promotes cell adhe-

sion and growth.

The ﬁrst culture of cells on microcarriers was developed by van Wezel

in the 1960s (van Wezel, 1967), using DEAE-Sephadex

50 gel beads

originally designed as ion-exchange chromatography beads. The ﬁrst

product to be produced industrially using microcarriers (an inactivated

polio vaccine) was developed by van Wezel himself several years later.

Nowadays, the main industrial use of microcarriers is in the production of

vaccines, but also of gene therapy vectors, recombinant proteins, and

mAbs. In the literature, there are reports of culture scale-up to 6000 L for

Vero cells cultivated on Cytodex

carriers (GE Healthcare, 2006). Figure

9.8 (see color section) shows Vero cells growing on microcarriers.

The materials used in the manufacture of microcarrriers are porous but,

depending on the pore dimensions, the carriers can be classiﬁed as

macroporous and microporous. The latter, also designated ‘‘solid’’ by

some authors, have diameters that are too small to allow cells to penetrate

the pores. Cell growth, in this case, is restricted to the carrier surface. On

the other hand, macroporous microcarriers, also simply designated ‘‘por-

ous’’ by some authors, have pores in the range of 10–400 m, allowing

cells to grow inside the pores. Generally, cell concentration inside or on

the porous matrices is in the range between 0.5 3 10

and 5 3 10

cells per

mL of microcarrier.

Most microporous microcarriers have a bead diameter of 90–300 m.

The cells grow on the surface of the beads, forming an attached mono-

layer, which may be susceptible to eventual collision of beads and to the

resulting mechanical damage. These microcarriers have densities of ap-

proximately 1.02–1.04 g cm

–3

, which is slightly higher than that of the

culture medium. Thus, they can be maintained in suspension at low

agitation speeds. Furthermore, since their diameter is relatively large, the

carriers settle down easily when agitation is stopped, facilitating harvesting

of cell-free supernatant.

In the case of macroporous microcarriers, they consist of particles in the

range of 0.4–5 mm, which present internal cavities that allow cell growth

inside the carriers. This type of microcarrier was developed with the

purpose of allowing tridimensional cell growth and attaining high cell

densities. The tridimensional growth favors the action of growth factors

produced by the cells themselves, decreasing the serum requirements and

facilitating the use of serum-free or protein-free media. The high cell

concentration makes the cell population more stable and increases culture

longevity, making macroporous microcarriers an interesting alternative for

long-term cultures. Additionally, their structure protects the cells from

shear damage in stirred systems.

There are several manufacturers of microcarriers for animal cell culture,

providing products with different chemical composition, size, form, and

density. Table 9.2 shows a list of common commercially available micro-

carriers and their manufacturers.

Bioreac tors for animal cells 229

When anchorage-dependent cells are immobilized by microcarriers they

can be cultivated in homogeneous bioreactors (Jo et al., 1998). Further-

more, the immobilization on the carriers allows the use of other bioreac-

tors that will be discussed in the following sections, such as ﬂuidized-bed

and packed-bed bioreactors. The microcarriers can be separated from the

supernatant by sedimentation, facilitating downstream processing or cell

retention in perfusion processes.

However, the use of microcarriers on a large scale presents some

problems regarding inoculum propagation. It may be desirable that cells

already attached to microcarriers used at an intermediate scale colonize

new carriers to inoculate a larger-scale culture. Enzymatic and sonication

methods can be used to release cells from microcarriers, but the conditions

can be harsh to the cells and present negative effects on cell physiology. A

more recent approach and an alternative to the total release of cells, is

bead-to-bead transfer. This technique seems to be highly dependent on the

combination of adhering characteristics of the cells and properties of the

microcarriers. The efﬁcient transfer of cells between particles is only

possible with some cell–carrier combinations (Wang and Ouyang, 1999).

Furthermore, the homogeneity of parameters and the mass transfer of

nutrients to the interior of macroporous microcarriers can be critical,

especially for larger beads and at larger cultivation scales (Rupp, 1987).

Packed-bed bioreactors

In packed-bed bioreactors, cells are maintained in a ﬁxed bed, that is,

conﬁned in a given environment. In most applications, cells adhered to

microcarriers are used in these bioreactors. Culture medium is recirculated

through the packed bed, promoting transfer of nutrients, metabolites, and

products. Before being pumped into the bed, the medium should be

enriched in dissolved oxygen. This can be accomplished by vigorous

stirring in aeration tanks or by the use of membrane modules, generally

positioned in an external recirculation loop. The size of the packed bed is a

critical parameter, since the dissolved oxygen concentration in the medium

Table 9.2 Commercially available microcarriers and their manufacturers

Product Material Manufacturer

Microporous microcarriers

Biosilon

Polystyrene with negative surface charge Nunclon

Cytodex

1 Cross-linked dextran with DEAE groups GE Healthcare

Cytodex

3 Cross-linked dextran coated with a denatured collagen

layer

GE Healthcare

HyQ

Sphere

Polystyrene with different options of charge and coating Hyclone/

SoloHill

Macroporous microcarriers

Cultispher

Cross-linked porcine gelatine Percell Biolytica

Cytoline

Polyethylene/silica GE Healthcare

Cytopore

Cross-linked cellulose with DEAE groups GE Healthcare

230 Animal Cell Technology

gradually decreases as the liquid ﬂows through the bed (Fassnacht and

Porter, 1999).

One of the advantages of this system is that a clariﬁed ﬂuid is obtained

at the bioreactor outlet, with no need for a cell separation device. Figure

9.9 (see color section) shows the operational scheme of a ﬁxed-bed

bioreactor and a photograph of a small-scale model.

Fluidized-bed bioreactors

Fluidized-bed bioreactors are also used mainly for the culture of cells

attached to microcarriers. However, in this case, microcarriers with a

higher density should be employed. In these bioreactors, a given amount

of microcarriers is ﬂuidized inside a compartment of the bioreactor,

avoiding cell damage due to bubble explosion. Figure 9.10 (see color

section) shows a photograph and an operational scheme of this type of

bioreactor.

These bioreactors are operated in perfusion mode and cell concentra-

tions reach 5 to 10 million cells per mL of medium. Several authors report

their results in terms of cell concentration per microcarrier volume and, in

this case, values in the order of 20 to 35 million cells per mL of carrier are

obtained (Wang et al., 2002).

Bioreactors providing surfaces for attached cell growth

Roller bottles and systems with multiple plates, such as Cell Factory (Nunc)

and CellCube

(Corning), provide a large surface area for cell adhesion and

proliferation. In the case of roller bottles, although the relative surface area

(area/volume) is small and consequently the production capacity is low,

large-scale plants can be established by using a large number of roller

bottles. This approach was adopted by the company Amgen for the

industrial production of erythropoietin. Operation costs for this type of

plant are high, because they are labor-intensive, and the risk of contamina-

tion is high, due to the manipulation of lots of small bottles.

On the other hand, the principle of CellCube

and Cell Factory is to

provide a large area for cell adhesion and proliferation in relatively

compact devices. The latter consists of multiple trays, providing surface

areas in the range of 700–25 000 cm

, for medium volumes of 0.2–8 L

(Nunc, 2006). The system called CellCube

provides a surface area in the

range of 8500–85 000 cm

and is ﬁtted with an automated control system

(Corning, 2006).

Hollow-ﬁber bioreactors

The core part of a hollow-ﬁber bioreactor is the hollow-ﬁber membrane

module, also simply known as the cartridge. It consists of a plastic cylinder

containing hundreds of semi-permeable capillary tubes, known as hollow

ﬁbers. The cells are inoculated in the extracapillary space (ECS). The cells

colonize the external surface of the ﬁbers and grow in this region. The

culture medium is pumped through the lumen of the ﬁbers, known as the

intracapillary space (ICS), as shown in Figure 9.11.

Bioreac tors for animal cells 231