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

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Amplified

heavy chain

cDNAs

Amplified

light chain

cDNAs

Lymphocytes from

immunized mouse

mRNA

Reverse transcriptase

cDNA

PCR PCR

Ligate H and L chain cDNAs

into expression vectorλ

H chain cDNA L chain cDNA

Package into phage

Combinatorial

phage library

Infect

H and L chains are

produced in infected cells

E. coli

H and L chains associate

to form Fab molecules

Prepare replica filter

Add labeled antigen

to filter

H and L chain-encoding cDNAs

from reactive antibody are cloned

Heavy chain

Light chain

Fab binds to filter

Fab binds

antigen

Isolate phage

DNA from

master plate

424 Animal Cell Technology

F(ab9)2 portion (Figure 17.4) normally last longer in circulation than do

smaller fragments (Fab, Fv, or scFv). If necessary, however, the half-life of

the smaller fragments can be increased by mixing them with a PEG, or by

chemical conjugation to form dimers, trimers, and tetramers (‘‘diabodies,

‘‘triabodies’’, and ‘‘tetrabodies,’’ respectively (Figure 17.4). These poly-

meric structures are obtained from the modulation of the length of the

ﬂexible peptide bridges of the scFv modules (Hudson and Souriau, 2003;

Roque et al., 2004).

It is also possible to produce bi-speciﬁc antibodies (Figure 17.4), which

have two variable regions, each with a distinct speciﬁcity, but which bind,

for example, to two adjacent epitopes of a given antigen, thus increasing

the avidity of the connection of the antigen to the antibody. Bi-speciﬁc

antibodies can also be generated so that they bind simultaneously to a

tumor antigen and a cytotoxic T lymphocyte, thus facilitating the elimina-

tion of the tumor (Hudson and Souriau, 2003). These bi-speciﬁc antibodies

can be produced by hybrid hybridomas (‘‘quadromas’’), by either chemi-

cal or genetic conjugation, as well as by the fusion of adhesive heterodimer

domains of two or more Fab modules (Roque et al., 2004).

Bi-functional antibodies can also be obtained from the fusion of natural

antibodies or recombinant antibodies with compounds that might carry

out auxiliary functions after antibody binding to its speciﬁc target (Hud-

son and Souriau, 2003; Roque et al., 2004). These compounds can be

radioactive conjugates, cytotoxic drugs, proteins, toxins, enzymes, and

viruses, any of which can have diagnostic or therapeutic uses.

17.5 Producti on systems

The development of a speciﬁc process for the production of an mAb

requires the selection of (a) a system of expression; (b) a bioprocess for

obtaining the product, (c) a puriﬁcation technique, and (d) analytical

methods for determining purity and product quality. It is clear that all the

choices must be compatible. So, for example, the selection of a process

must be compatible with the expression system. Moreover, the process

chosen must consider the speciﬁc conditions of the product in relation to

the competitive market, the quality required, and the total volume to be

produced.

Thus, for diagnostic or biogeneric industries, the production cost

dominates the choices, and the optimization of the productive process is

extremely important (NAS, 1999). This is not true for the therapeutic

industry, or for products protected by patents, which are more dependent

on the regulatory requirements.

Unlike the production of other proteins from animal cells, mAbs can

also be produced in vivo by inducing ascitic tumors in laboratory animals,

Figure 17.5 (opposite)

Construction of a mAb combinatory library expressed in a bacterial system (from

Recombinant DNA, 2/e by James D. Watson, et al. # 1992 by James D. Watson,

Michael Gilman, Jan Wirkowski, and Mark Zoller. Used with permission).

Monoclonal antibodies 425

as described above. This is an option when using hybridomas (Falkenberg,

1998; Hendriksen and Leeuw, 1998). However, there is a drive worldwide

to ﬁnd suitable in vitro production methods, to avoid the suffering of

laboratory animals, as well as minimizing the risks of contamination of the

ﬁnal product by adventitious substances. However, in certain speciﬁc

situations, the use of an in vivo system of production is unavoidable. This

includes the following: (a) when the mAb concentrations are low

(, 5 g/ml in batch systems, 50  g /ml in hollow-ﬁber bioreactors, or

300 g/ml in membrane bioreactors, (b) when it has been proved that the

use of cell culture results in a loss or decrease of the speciﬁc function of

the mAb; (c) when the hybridoma line can only grow and synthesize the

product in vivo; and (d) when the quantity to be produced exceeds the

laboratory capacity of the producer (NAS, 1999). A noteworthy example

of such a product generated in vivo is the therapeutic agent OKT3

, which

can lose its biological activity when puriﬁed from the supernatant of a cell

culture.

For antibodies produced in vitro, as is the case for many other proteins

obtained from animal cell cultures, the main question involves the low

level of expression of these products in the culture medium. This necessi-

tates the use of large culture volumes for production, thus involving higher

costs, especially for puriﬁcation. In general, the optimization of these in

vitro processes attempts to increase the concentration of the product in the

medium. This is often possible by using high cell densities. Typical values

for traditional processes are in the range of 20–100 mg/L, whereas for

optimized systems, this can rise to 4.6 g/L (Kretzmer, 2002; Wurm, 2004).

It is unusual to ﬁnd an optimized generic process for obtaining mAbs

since each producer cell has a unique pattern of response to stress,

consumption of nutrients and synthesis of products and byproducts.

However, it is clear that the systems utilized for the production of mAb

do not differ in any signiﬁcant way from those using animal cells for the

synthesis of other products, as can be seen in various chapters of this book

(Chapters 5, 9, 11, and 12). For this reason, only those aspects that are

especially relevant for obtaining mAbs from in vitro systems will be

presented here.

17.5.1 Cell lines

Although various systems have been developed for the production of

mAbs, only those involving cultures of animal cells will be presented in

this chapter, since this is the system used to produce most of the antibodies

on the market today (van Dijk and van de Winkel, 2001). Those of murine

origin are produced directly from hybridomas, whereas the humanized or

completely human ones come from the culture of animal cells transfected

with speciﬁc genetic sequences, which are capable of following the

patterns of glycosylation and the desired structural conformation, required

for adequate drug performance.

Hybridomas constitute the most widely used cell lines for the produc-

tion of mAbs, on both small and large scales. Section 17.4 of this chapter

presents details of methods used to construct this type of cell. However,

these cells produce antibodies that have limited therapeutic application,

426 Animal Cell Technology

due to the immunological responses to murine antibodies that often arise

in patients.

As discussed above, one solution for this problem is the genetic

manipulation of cell lines to enable them to synthesize humanized anti-

bodies. In this situation, the hybridomas serve as an important source of

gene sequences that codify antibody molecules (or their fragments),

although these are later transfected into other animal cells. In general, the

cell lines used are more robust than hybridomas, due either to their greater

stability or even a reduced tendency for apoptosis.

The Chinese hamster ovary (CHO) cell line is now being used as a

standard host cell for transfection with genes of interest for later use in the

production of recombinant antibodies (Butler, 2005). Other cells with

equivalent performance are the murine myelomas NS0 and Sp2/0, as well

as baby hamster kidney (BHK), human embryonic kidney (HEK-93), and

a derivative of the human retina (PER.C6

) (Chu and Robinson, 2001).

The selection of the best cell line should consider antibody productivity,

as well as cell growth rate, although these parameters frequently follow

opposite trends (Wurm, 2004). For the production of large amounts of

mAb, it is fundamental that the productivity of the selected cell line is

high. Otherwise, larger reaction volumes will be required, and the cost of

puriﬁcation will be increased. A reference value for speciﬁc protein

productivity is 20 pg/cell per day.

Another important aspect involved in the selection of transfected lines is

the capacity to grow without physical support, since the scale-up of such

processes is much simpler than those designed for growth of anchorage-

dependent cells. Thus, cells that grow naturally in suspension are pre-

ferred, such as myeloma cells (Sp2/0 and NS0), or others that can be easily

adapted to this form of cultivation, such as CHO and BHK (Chu and

Robinson, 2001).

17.5.2 Basic conditions for in vitro cultivation

Culture medium

Traditionally, the production of mAbs uses complex culture media con-

taining glucose and amino acids as the main sources of carbon for cell

metabolism, as well as vitamins, micronutrients and sometimes animal

serum, usually fetal bovine serum. Chapter 5 provides a discussion on

composition of culture media and recent trends in the search for formulas

that do not require the use of animal serum, or of proteins of animal

origin. These serum-free formulations use substitutes such as peptones,

epithelial and ﬁbroblast growth factors, hydrolysates, yeast extract, cho-

line, and inositol. For the production of mAbs, various serum-free

formulas are available, some of these developed speciﬁcally for a given cell

line (Chu and Robinson, 2001). The development of those media is easier

for non-anchorage-dependent cells, such as those used for mAb produc-

tion. Thus, approximately 50% of the antibodies for therapeutic use are

already produced using serum-free media. In some circumstances, the

elimination of serum should be accompanied by the addition of other

substances with the same shear stress protective effect of serum proteins,

Monoclonal antibodies 427

such as Pluronic

F68 and carboxymethylcellulose (CMC) (Wu, 1995;

Chu and Robinson, 2001).

Most of the cell culture media are formulated to attain a physiological

osmolality in the range of 270–330 mOsm/kg. Under hypo-osmotic stress,

hybridomas show a reduction in cell growth rate without any speciﬁc

increase in antibody productivity (Ryu and Lee, 1999). However, under

hyperosmotic stress, some hybridomas show physiological changes such

as an increase in cell size, reduction in speciﬁc growth rate, and increase in

speciﬁc mAb synthesis (Cherlet and Marc, 2000). The overall effect of

these changes is a function of the cell line, and may or not result in an

increase in the production of the antibody (Ozturk and Palsson, 1991;

Bibila et al., 1994). The addition of osmoprotectors such as glycine, betain,

sarcosine, glycine, and proline may attenuate the toxic effects of high

osmolality on growth, with an overall positive effect.

Inﬂuence of oxygen

Oxygen is crucial for the production of energy during phosphorylation

and for the synthesis of cell components. In most cases, adequate growth

conditions require an excess of dissolved oxygen (DO), with the optimum

dependent on the cell line, cultivation conditions, and growth phase. The

critical range of DO, that is, that which limits growth and/or mAb

production, also varies as a function of cell line and cultivation conditions,

and usually is in the range of 10–20% of air saturation.

The best system for aeration and stirring of culture medium in a

bioreactor must minimize shear stress without signiﬁcantly reducing the

oxygen transfer (K

a) and yet avoiding the generation of foam. In some

systems, protectors such as serum, Pluronic

F68, PEG, dextrans, lipids,

and cholesterol, may have to be used to prevent shear stress. Sensitivity to

shear depends on the cell line, and fortunately the lines most often utilized

industrially for the production of mAbs, such as hybridomas, CHO, and

BHK-21, are among the most resistant animal cells in relation to these

hydrodynamic forces (Chisti, 2000; Chu and Robinson, 2001; Wu, 1995).

17.5.3 Cell metabolism

As discussed in Chapter 4, glucose and glutamine are the two main

substrates in culture media used in animal cell processes, for generating

intermediate components of anabolism and catabolism (Doverskog et al.,

1997). The fraction of the energy produced by the metabolism of each of

the substrates depends on the cell line. Under conditions of excess glucose,

the hybridomas normally obtain 90% of the ATP necessary for metabolic

functions from glutamine, whereas CHO cells only obtain 40% (Jeong

and Wang, 1995).

An excess of glucose and amino acids, especially glutamine, generates

lactate and ammonia, respectively, and in many cases, these compounds

inhibit the growth of animal cells and the formation of the products of

interest, although the rate of mAb formation is not necessarily affected

(Schneider et al., 1996; Doverskog et al., 1997). Large quantities of these

metabolites in the culture medium can also diminish the quality of the

428 Animal Cell Technology

ﬁnal product. Some cells can eliminate some of the metabolites by excret-

ing them in the form of alanine, which is a non-toxic byproduct.

Lactate reduces the internal pH of the cells or of the culture medium, as

well as increasing the osmolality of the medium. The mechanisms of

toxicity of ammonia are less well understood (Schneider et al., 1996). The

critical concentrations of these compounds depend on the cell line and the

culture conditions. Typical critical values for ammonia in the production

of mAbs are in the range of 2–10 mM (Schneider et al., 1996), whereas for

lactate the range is much broader, varying from 1 to 300 mM.

17.5.4 Bioreactors and operation mode

Various combinations of bioreactors and operation mode have been used

for the production of mAbs in several systems of expression, as shown in

Chapter 9. All cells utilized for the production of mAbs grow in suspen-

sion. Those that did not initially have this capacity have been adapted (as

is the case for CHO and BHK) (Butler, 2005). This results in a large

number of options for production systems. Cells with this characteristic

are easily cultivated in stirred-tank reactors, which have been scaled up to

a volume of 10 000 L (Chu and Robinson, 2001; Kretzmer, 2002). This

kind of bioreactor provides excellent homogeneity, facility for the imple-

mentation of control techniques, and the principles of scaling up are

relatively well known. Other kinds of bioreactors for the production of

mAbs are also available, such as air-lift, with volumes up to 1000 L, and

also ﬁxed-bed bioreactors (Moro et al., 1994; Irving et al., 1996; Kretzmer,

2002).

Products with less demand, such as those used in diagnosis, are devel-

oped in small-scale systems such as T-ﬂasks, rollers, and hollow-ﬁber

bioreactors (Kretzmer, 2002). The reduced size of these production

systems makes it possible to operate various units in parallel to obtain

different products. A small increase in scale can be reached by the multi-

plication of units.

Hollow-ﬁber bioreactors constitute an optimized production system

where it is possible to achieve higher cell concentrations (10

to 10

cells/

ml), and the product concentration can reach a level of 0.7–2.3 g/L, which

is similar to what can be obtained with ascitic ﬂuid (Hendriksen and

Leeuw, 1998). This system can operate for over 3 months without affecting

cell viability, but presents problems with mass transport, and the forma-

tion of nutrient gradients, which require speciﬁc solutions (Kretzmer,

2002).

The operation mode used in the industry is predominantly batch, given

the simplicity and facility of control, good reproducibility, lower indices

of contamination, and low production costs (Xie and Wang, 1997). How-

ever, this operation mode has some disadvantages, the low cell and product

concentration, because it permits the accumulation of ammonia and

lactate, or the exhaustion of essential nutrients (Xie and Wang, 1997,

2006). Typical antibody concentration values in a batch system using a

stirred-tank bioreactor or air-lift are in the range of 100 g/ml.

Two approaches are possible to maximize the concentration of anti-

bodies in the culture. The ﬁrst is based on an increase in the speciﬁc

Monoclonal antibodies 429

production rate by adding epidermal or ﬁbroblast growth factors, IL-2

(Jang and Barford, 2000), butyric acid (Cherlet and Marc, 2000), or cyclic

nucleotides (Dalili and Ollis, 1988). Moreover, the addition of thymidine,

the reduction or elimination of serum, suboptimal concentrations of oxy-

gen, a reduction in pH, and the increase in osmolarity are situations that

can disturb growth, resulting in an increase in the speciﬁc antibody

production rate (Ozturk and Palsson, 1991; Reddy et al., 1992).

The second focus is based on obtaining greater cell concentrations for

longer time periods to maximize the production of mAbs (Duval et al.,

1991; Bibila and Robinson, 1995). In general, at least one of the following

factors would discourage growth and/or production: (a) exhaustion of

nutrients, especially certain essential amino acids, vitamins, glucose or

serum (Glassy et al., 1988; Duval et al., 1991; Jo et al., 1993a, 1993b; Hiller

et al., 1994); (b) inhibition due to the formation of toxic byproducts, such

as lactate and ammonia (Ozturk et al., 1992); and (c) inadequate concen-

tration of dissolved oxygen. Satisfactory results can be obtained simply by

increasing the concentration of nutrients and promoting an equilibrium of

salts to guarantee adequate osmolality (Jo et al., 1993a). However, there is

no way of avoiding the constant accumulation of inhibiting metabolites,

which ends up leading the process to collapse.

The fed-batch operation mode has been widely used in the industry due

to the simplicity of operation and control possibilities, easy scaling-up,

and efﬁciency in obtaining high cell and product concentrations (Jo et al.,

1993b; Bibila and Robinson, 1995; Xie and Wang, 1997; Sauer et al., 2000).

The increase in productivity is due to the manipulation and exploration of

the physiological state of the cells, redistributing the available resources

and rerouting the ﬂow of metabolites. This avoids the synthesis of toxic

byproducts and, consequently, prolongs the phase of product accumula-

tion. However, it is inevitable that a progressive reduction in viability will

arise from problems of accumulation of toxic byproducts and high

osmolality.

The continuous process is the only way of avoiding the accumulation of

inhibitors, since it guarantees their constant removal at the same time as it

supplies the system with the nutrients necessary for the performance of

the cells. If perfusion is used, with cell retention, the system provides a

high cell density that is controllable, without gradients, and which can

maintain optimal conditions of nutrient concentration and toxic by-

products.

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Monoclonal antibodies 433