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

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The ﬁbers can be made of different materials, such as cellulose esters

and polysulfone. The total surface area of a hollow-ﬁber bioreactor varies

in the range of 0.5–3.5 m

. The pore size of ﬁbers commonly employed in

animal cell culture corresponds to a molar mass cut-off between 10 and

100 kDa.

The porous nature of the ﬁbers allows for exchange of nutrients and

metabolites. Low-molar-mass molecules, such as glucose and ammonia,

can move freely through the pores of the ﬁbers, at a rate that is controlled

just by the pressure gradients generated by the medium recirculation

pump. High-molar-mass proteins, which can be produced by the cells or

added as nutritional supplements to the extracapillary space, are not able

to permeate the membrane ﬁbers and are retained in the cell bed in the

ECS.

Thus, the cell population grows in the ECS of the hollow-ﬁber car-

tridge, receiving the nutrients originating from the culture medium that is

recirculated through the interior of the ﬁbers. Inhibitory metabolic pro-

ducts, generated as a consequence of cell growth, diffuse in the opposite

direction and get diluted in the recirculating culture medium. In the same

way, low-molar-mass proteins and growth factors produced by the cells

or provided by serum can permeate the ﬁbers. Thus, in the axial direction

of the ﬁbers a decreasing gradient of medium components and an increas-

ing gradient of metabolites are formed. This process may lead to a decrease

in the transfer of nutrients to the microenvironment of the cells, with the

possibility of the concentration of these molecules becoming growth-

limiting. This may become critical when ﬁber length is increased, since a

region very poor in nutrients and very rich in inhibitory metabolites can

be created in the ﬁnal portion of the ﬁbers. This situation can be

attenuated through the introduction, directly through the ECS, of an

additional stream perpendicular to the ﬁbers, known as recycle stream.

Culture medium containing serum can be added to this recirculation

stream, while basal medium is recirculated through the lumen of the ﬁbers.

Figure 9.12 shows the scheme of a hollow-ﬁber bioreactor ﬁtted with an

extracapillary recirculation line.

Medium

inlet

Medium

outlet

ECSICS

Figure 9.11

Simpliﬁed representation of a hollow-ﬁber module with only three ﬁbers. The

straight arrows pointing from the intracapillary space (ICS) to the extracapillary

space (ECS) represent the transfer of nutrients from the culture medium to the

cells (inoculated in the ECS), whereas the straight arrows in the opposite direction

represent the transfer of metabolites and products from the cells to the ﬁber

interior.

232 Animal Cell Technology

Oxygen transfer is an important issue in the operation of a hollow-ﬁber

bioreactor. In these bioreactors, a membrane-based aeration system is

usually included in the intracapillary recirculation loop to enrich the

recirculating culture medium with oxygen. However, due to the low

solubility of oxygen in aqueous solutions, the recirculation speed through

the ﬁbers must be very high.

It is possible to modify some design parameters to determine optimal

operational conditions for these bioreactors, such as the ﬁber material, the

packing density of ﬁbers inside the module, the dimensions of the module,

the pore size of the ﬁbers, and the type of aeration system used in the

recirculation loop. Furthermore, the yield of these bioreactors can also be

improved by optimizing the operational strategies, such as: implementa-

tion of an extracapillary recirculation loop; increase of the ﬂow rate

through the oxygenation device; execution of periodic cell purges from the

ECS, to avoid culture necrosis; use of an adequate serum content or of an

improved medium composition to favor cell proliferation; and determina-

tion of an optimal time and scheme for the extracapillary recirculation.

Parameters that are traditionally used to evaluate the metabolic activity

of the cell population inside a hollow-ﬁber bioreactor include glucose

consumption rate (r

GLC

) and oxygen consumption rate (r

). However,

these parameters are highly dependent on variations in nutrient feed and

metabolic products removal (Gramer et al., 1999). The use of a strategy to

overfeed the ICS with nutrients results in a rapid increase in r

GLC

. On the

other hand, an increase in product titer and an improved metabolic

Residue

Basal medium

Oxygenator

Serum-containing medium

Product harvesting

Recirculation

pump

Pressure sensor

Intracapillary circuit

Extracapillary circuit

pH sensor

Extra-

capillary

chamber

Pressure sensor

Hollow-

fiber

cartridge

Intra-

capillary

chamber

Figure 9.12

Schematic representation of a hollow-ﬁber bioreactor ﬁtted with an extracapillary recirculation

stream.

Bioreactors for animal cells 233

efﬁciency in terms of product formation can be attained if an operation

strategy is adopted that includes a perpendicular extracapillary recircula-

tion stream and a regular purge of cells, to operate at lower biomass

concentrations (Rodrı´guez et al., 2005).

In a study of the production of a humanized mAb produced by NS0

cells, Rodrı´guez et al. (2005) compared two different operation strategies

(Figure 9.13). The optimized strategy provided a signiﬁcant decrease in the

ratio of the ﬂow rates of product harvest and medium feed, with a twofold

increase in the total amount of antibody harvested and a sixfold decrease

in the harvested volume. Thus, antibody concentration in the product

stream was increased by over 10-fold, with a positive impact on the

puriﬁcation process of this mAb.

9.4 Modes of operation of bioreactors

The different operation modes used in microbial fermentations are em-

ployed also in animal cell cultivation. Although many different classiﬁca-

tions can be adopted, the most general is the one that considers the

following operation modes: batch, fed-batch, continuous, and perfusion,

which is a continuous mode with cell recycle/retention (Castilho and

Medronho, 2002).

The behavior of a culture system in terms of cell, nutrients, and product

concentrations can be described mathematically by mass balances and

200

400

600

800

Intracapillary recirculation

flow rate (mL h )

⫺1

Basic operation

Optimized strategy

0.0

0.2

0.4

0.6

0.8

Ratio of product harvest flow

rate and extracapillary

recirculation flow rate

r (mmol h )

GLC

⫺1

0010 1020 2030 3040 4050 5060 60

Time (days)

Antibody produced (g)

Figure 9.13

Cultivation of NS0 cells in an AcuSyst Jr

hollow-ﬁber bioreactor: comparison of

two different operation strategies, designated basic and optimized strategy

(Rodrı

guez et al., 2005).

234 Animal Cell Technology

kinetic expressions, such as the Monod equation, which describe the

effects of nutrient and metabolite concentrations on the speciﬁc reaction

rates.

In order to make a decision on the most appropriate operation mode for

a bioreactor, several factors must be taken into consideration. At industrial

scale, the most important factors are:

(i) Characteristics of the cell line used, such as the stability of expression

of the product, production pattern, degree of resistance to inhibitory

metabolites, and resistance to shear stress;

(ii) The market demand for the product, which determines the produc-

tion scale;

(iii) The technical experience of the teams responsible for both process

development and regulatory issues.

9.4.1 Batch cultivation

Batch is a discontinuous operation mode, and cell growth occurs without

any additional supplementation of nutrients after inoculation of cells.

While substrates are metabolized, the cell population grows, forming the

product and other metabolites. The volume is maintained constant

throughout the whole process.

Equations 1 to 3 represent the generic mass balances for cells, substrate

and product in a batch cultivation process:

¼ X

(1)

¼q

(2)

¼ q

(3)

Where:

¼ viable cell concentration;

t ¼ cultivation time;

 ¼ speciﬁc growth rate;

S ¼ concentration of a generic substrate;

¼ speciﬁc consumption rate of a generic substrate;

P ¼ concentration of a generic product;

¼ speciﬁc formation rate of a generic product.

Figure 9.14A shows the operational conﬁguration and typical proﬁles

for cell and substrate concentration in a batch culture. Figure 14B shows

the concentration of cell, product (mAb), and substrates (glucose and

glutamine) for a myeloid transfectoma in batch cultivation.

In a batch cultivation, cell concentration increases from the inoculation

density (0.1–0.4 million cells per mL) up to maximum values between 2

and 4 million cells per mL, being the upper limit applicable to insect cell

cultivation. Since animal cells present speciﬁc growth rates between 0.02

and 0.04 h

–1

, the time period to attain maximum cell density in a batch

Bioreac tors for animal cells 235

culture is approximately 5 days. The range of speciﬁc product formation

rates is wider, varying from a value as high as 30 pg cell

–1

day

–1

, reported

for mAb production by transfectomas (Velez et al., 1987), to values two

orders of magnitude lower (Altamirano et al., 2004). The concentration of

a secreted product in batch cultures is usually in the range of 1–50 mg L

–1

After attaining maximum product concentration, the culture is interrupted

and the spent medium is sent to the downstream processing stages.

Due to the low solubility of oxygen, this gas must be supplied con-

tinuously, instead of just at the start of the culture, as in the case of the

other nutrients. Control of pH is carried out through addition of base and

by varying the CO

concentration in the gas phase, since most animal cell

culture media contain sodium bicarbonate.

0.0E 00⫹

1.0E 06⫹

2.0E 06⫹

3.0E 06⫹

Viable cells Product Glucose Glutamine

(A)

(B)

Time

Cell concentration (cells/mL)

0.0 2.5

5.0 7.5

10.0

Time (days)

Product concentration (mg/L),

glucose and glutamine (mM)

Figure 9.14

Batch cultivation: (A) operational conﬁguration and typical viable cell (Xv) and

substrate (S) concentration proﬁles; (B) concentration of cells, product and two

substrates in a batch bioreactor culture of a myeloid transfectoma producing a

humanized monoclonal antibody (Center of Molecular Immunology – CIM, Cuba).

236 Animal Cell Technology

The batch operation mode is the simplest to carry out, and therefore it

is widely employed. Generally, it is adopted for the cultivation of cells in

stationary ﬂasks, in stirred ﬂasks, in small- and intermediate-scale bioreac-

tors for inoculum propagation and also in several industries at production

scale. In the case of inoculum propagation, the culture is stopped when the

cells are still in the exponential growth phase, to provide a high mass of

cells growing at maximum rate. In this way, it is guaranteed that these

cells, when inoculated in a larger bioreactor, will present a minimal lag

phase, and so reducing the non-productive period.

The low volumetric productivities that characterize batch cultivation

processes are a disadvantage for the use of this operation mode for

production. However, a variant known as ‘‘repeated batches’’ is an inter-

esting alternative. It consists of initially carrying out a batch cultivation

for the time needed to attain the desired product concentration. At that

moment, just a part of the bioreactor contents is harvested. The remaining

cell suspension inside the bioreactor is then used as inoculum for a new

batch, by ﬁlling the vessel with fresh medium. This procedure can be

repeated several times, until a decrease in cell growth or product formation

is observed. The use of repeated batches allows a decrease of the time the

bioreactor is non-productive. This eliminates the time periods that would

be necessary for cleaning and sterilization between each batch.

The productivity of batch cultures in general is limited by the low cell

concentration and, consequently, the low product concentration at the end

of the process. This is related to the fact that the initial concentration of

substrates must be relatively low to avoid cell death due to osmotic effects

(Morbach and Kra

mer, 2003), as well as to the fact that the cells are

submitted to environmental conditions that keep changing during the

whole process.

The main technological features of the batch operation mode are:

(i) operational simplicity;

(ii) simple equipment and operational procedure, decreasing contamina-

tion risks, when compared with other operation modes;

(iii) low productivity, that can be improved slightly by carrying out

repeated batches;

(iv) process optimization possible through manipulation of medium com-

position and determination of the best time to harvest the product;

(v) applied in industrial processes at scales up to 20 000 L.

9.4.2 Fed-batch cultivation

The difference of this operation mode compared with batch is that one or

more nutrients are fed during cultivation, to replace those consumed by

the cells. Fed-batch cultures begin with a working volume lower than the

maximum working volume, so that nutrients can be added during the

culture period, either from fresh culture medium or from a concentrated

nutrient solution. Figure 9.15A shows typical proﬁles for cell and product

concentrations, as well as the volume of medium in the reactor resulting

from pulse additions of nutrients.

Bioreac tors for animal cells 237

Feeding nutrients during the culture allows an increase of the total

amount of nutrients metabolized by the cells, which results in higher ﬁnal

concentrations of cells and product. Effects of high initial nutrient concen-

trations can be eliminated, since the nutrients are not added all at once at

the beginning of the process.

Equations 4 to 6 present the mass balances for cells, substrate, and

product in a fed-batch culture:

¼ X



(4)

¼q

 S) (5)

0.0E 00⫹

1.0E 06⫹

2.0E 06⫹

3.0E 06⫹

4.0E 06⫹

5.0E 06⫹

100

120

Viable cells Product Glucose Glutamine

(A)

Time

(B)

Cell concentration (cells/ml)

0.0 2.5

5.0 7.5

10.0

Time (days)

Product concentration (mg/L),

glucose and glutamine (mM)

Figure 9.15

Fed-batch cultivation: (A) operational conﬁguration and typical volume, cell, and

substrate proﬁles, for a pulse feeding strategy; (B) concentration of cells, product,

and two substrates in a fed-batch bioreactor cult ure of a myeloid transfectoma

producing a humanized monoclonal antibody (Center of Molecular Immunology –

CIM, Cuba).

238 Animal Cell Technology

¼ q



P (6)

Where:

F ¼ feed ﬂow rate, which usually varies with time;

¼ concentration of a generic substrate in the feed stream;

V ¼ bioreactor working volume, which varies with time.

As can be seen, these equations differ from those presented for batch

cultures, since they contain terms related to the addition of nutrients from

a solution with concentration S

at a ﬂow rate F.

Figure 9.15B shows the behavior of a fed-batch culture. Starting from

the inoculum concentration, a cell density of 5 3 10

cells mL

–1

is obtained

within a period of 7–10 days, although in the literature higher concentra-

tions (10 3 10

cells mL

–1

) have been reported (Xie and Wang, 1997).

Furthermore, in some cases, the cultivation time can be extended to 10–15

days. The most relevant in this context is that product concentration can

be increased signiﬁcantly, when compared with batch cultivation. This is

evidenced by Equation 6 (mass balance for product), taking into consid-

eration that values for cell mass and culture times are larger in this case

than in batch cultures. Indeed, some of the ﬁnal concentration values

reported for this operation mode are among the highest titers attained for

animal cell products. Conventional product concentrations of 150 mg L

–1

are usually achieved in fed-batch cultures, but after optimization of

feeding strategies and using improved recombinant cell lines, Robinson

(1994) reported product concentrations of approximately 2 g L

–1

Nutrient feeding can be carried out according to different strategies;

addition of nutrients in pulses, in steps, at constant ﬂow rate, or using

more sophisticated strategies, such as an exponentially growing feed ﬂow

rate. By using optimized strategies, based on the speciﬁc consumption

rates for each substrate and on the effect of nutrient concentration on these

rates, cell concentrations up to 2.5-fold higher than those observed in

batch mode were achieved by Xie and Wang (1997).

Another interesting positive effect that has been observed in fed-batch

cultivation is the duration of the stationary phase (Altamirano et al., 2004).

When cells show non-growth associated or inversely associated product

formation kinetics, the prolonged stationary phase observed in fed-batch

cultures results in considerable gains in productivity. The use of molecular

biology tools to change the production pattern of a cell line for a non-

growth associated pattern aims at exploiting this advantage (Weikert et al.,

1999).

In fed-batch culture processes, productivity is usually limited by the

accumulation of toxic metabolites, which can inhibit cell growth and

negatively inﬂuence culture viability and product formation. The most

relevant toxic metabolites are lactate and ammonia (Hassell et al., 1991).

Feeding strategies aimed at maintaining low levels of nutrients, as well as

total or partial substitution of glucose and glutamine by slowly metabo-

lized substrates, allow a decrease of the level of lactate and ammonia in

cultures. In a fed-batch culture of a tPA-producing CHO cell line,

Altamirano et al. (2004) proposed replacing glucose by galactose, a more

Bioreac tors for animal cells 239

slowly metabolized sugar, and this caused a decrease in the generation of

lactic acid, as well as an increase in cell viability and product formation.

Another active research ﬁeld is the optimization of intracellular meta-

bolic reactions to decrease the production of toxic metabolites through

metabolic engineering strategies. An example is the introduction of the

glutamine synthetase (GS) gene into NS0 cells, that do not have this

activity, or into CHO cells, that have a low endogenous GS activity.

GS catalyzes the formation of glutamine from glutamate, allowing cells

to grow in a medium with low concentrations of glutamine or no

glutamine at all. The introduction of this gene leads to a considerable

decrease in ammonia levels in the cultures, resulting in a signiﬁcant

increase in maximum cell concentration and prolonged duration of the

stationary phase. As a consequence, ﬁnal product concentration and

volumetric productivity are increased (Bebbington et al., 1992). This

approach has been applied industrially for both cell lines – NS0 and

CHO.

A disadvantage of fed-batch culture is the long residence time of the

product in the culture environment. A product molecule, secreted at the

early phase of the process, remains inside the bioreactor for the whole

duration of the culture, in the presence of proteases and glycosidases

(Goochee and Monica, 1990). Since the product of interest is generally a

protein (usually a glycoprotein), these enzymes may degrade the product

and, in some cases, destroy an important fraction of the active material

that is synthesized. For that reason, molecules that are unstable at the

culture temperature or prone to enzymatic cleavage should not be pro-

duced by fed-batch cultures.

The most important characteristic of the fed-batch mode is the fact that,

in spite of its more complex operation compared with batch cultures, the

productivity is increased signiﬁcantly. Furthermore, when compared with

perfusion operation, the runs are considerably shorter, making process

validation easier. For this reason, a relatively large number of biotechnolo-

gical companies have developed their production processes based on this

technology (Chu and Robinson, 2001).

In summary, the main technological features of this operation mode are:

(i) moderate operational complexity;

(ii) moderate contamination risks;

(iii) intermediate volumetric productivity;

(iv) long residence time of product in the bioreactor;

(v) process optimization possible basically through manipulation of

medium composition, feeding strategy, and harvesting time;

(vi) applied in industrial processes at scales up to 20 000 L.

9.4.3 Continuous cultivation

Continuous cultivation is used mainly in research and development

activities, at small scales. It is characterized by a continuous feed of fresh

medium and a continuous removal of cell suspension, both at the same

ﬂow rate, and at constant bioreactor volume.

240 Animal Cell Technology

Equations 7 to 9 describe the behavior of a continuous culture:

¼ X

 DX

(7)

¼q

þ D(S

 S) (8)

¼ q

 DP (9)

Where:

D ¼ dilution rate (D ¼ F/V).

When the system reaches the steady state, concentrations of cells,

product, and substrates remain constant, so that according to Equation 7

the speciﬁc growth rate becomes equal to the dilution rate.

Figure 9.16A shows a typical operational conﬁguration of a continuous

bioreactor culture. In Figure 9.16B, the cell concentration data of a real

continuous culture, operated at three different dilution rates, are pre-

sented. The rectangles indicate the different steady states observed,

whereas the arrows show the moment of change in dilution rate. Further-

more, a guide line has been included in Figure 9.16B to help interpretation

of the cell concentration proﬁle.

The continuous mode allows the establishment of well-deﬁned steady

states, so that the relationship between the concentration of substances

inside the bioreactor and different biological reaction rates can be studied.

Therefore, this operation mode is a powerful tool for cell characterization

(Altamirano et al., 2001).

When carrying out studies in continuous mode, it is important to

guarantee that the system stabilizes before measurements are made, to

ensure that reliable results are obtained. The determination of physiologi-

cal parameters in non-steady state can be inﬂuenced by effects of adapta-

tion or transition of the biological system, therefore making measurements

imprecise (Vits and Hu, 1992).

A limiting condition when carrying out continuous culture is the

situation known as bioreactor wash-out. This condition is characterized

by a cell removal rate at the bioreactor outlet that is equal to or higher

than the cell growth rate, such as that shown in Figure 9.16B after D has

been increased to 0.8 d

–1

. The wash-out condition occurs when the

dilution rate is increased to a threshold value called D

wash-out

, which can be

derived from Equation 7, considering steady state and adopting 

MAX

for .



MAX

¼ D

washout

(10)

Dilution rate values higher than D

wash-out

cause a progressive decrease in

cell concentration, until no more cells are found inside the bioreactor.

Thus, D

wash-out

is the upper limit that should not be exceeded in a

continuous culture. Since the 

MAX

values for animal cells are between

0.02 and 0.04 h

–1

(0.5–1 d

–1

), the maximum cell concentration usually does

not surpass 2 3 10

cell mL

–1

. Considering that productivity in a contin-

Bioreac tors for animal cells 241