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

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16.4.2 Characterization of biopharmaceuticals

Biopharmaceuticals have completely different properties from conven-

tional low molar mass pharmaceuticals. This particularly affects product

characterization, due to the complex production processes and protein

structures. Thus, successful production of biopharmaceuticals relies

mainly on strict protocols, clinical expertise, and follow-up during clinical

application (Crommelin et al., 2003).

For these reasons, the ability to compare products originating from

different processes is more important than characterizing a biopharma-

ceutical product from a well established process. Modiﬁcations in the

production process can result in changes in physicochemical properties

and post-translational modiﬁcations. These modiﬁcations can inﬂuence

biological activity, bioavailability, and immunogenicity of the product

(Chirino and Mire-Sluis, 2004). Therefore, as discussed in Chapter 14, the

regulatory agencies worldwide are discussing criteria to be applied to

evaluate comparability of biological products (Food and Drug Adminis-

tration, 2005; European Medicines Agency, 2005).

Among the many tests available for characterizing biopharmaceuticals,

assessment of protein structure, particularly quaternary structure, includ-

ing aggregate formation, seems to be particularly important, since it can

strongly inﬂuence immunogenicity. This remains a concern for the char-

acterization of biological products, since preclinical studies, carried out in

animals, are not able to predict the immunogenicity of a biological drug in

the target population (Chirino and Mire-Sluis, 2004). In this context,

product labelling becomes an important issue, since aggregate formation

and continuous administration of a protein drug can lead to antibody

production (Ryff and Schellekens, 2002). Biopharmaceutical labelling

could thus provide information in such a way that the physician would

understand the clinical and biological consequences of antibody produc-

tion and, therefore, could make a decision that could lead to the suspen-

sion of a treatment.

16.4.3 Alternative expression systems

Due to the structural complexity of therapeutic proteins, their recombi-

nant forms are usually produced in mammalian cell culture. When glyco-

sylation is not present or is not essential for biological activity, therapeutic

proteins can be produced in bacteria (e.g. E. coli) or yeast (e.g. Sacchar-

omyces cerevisiae). However, some studies propose the use of these

systems for glycosylated proteins as well.

Baneyx and Mujacic (2004) propose the genetic manipulation of E. coli,

to enable metabolic competency to carry out post-translational modiﬁca-

tions. Gerngross (2004) argues for the use of ﬁlamentous fungi and yeast

for the production of therapeutic proteins by the expression of human

glycosylation enzymes. This type of strategy was reported by Chiba et al.

(1998) and Martinet et al. (1998) for S. cerevisiae and Pichia pastoris,

respectively.

Plant cells are another expression system, which is being evaluated for

the production of recombinant therapeutic proteins. According to Hellwig

404 Animal Cell Technology

et al. (2004), recombinant human glycoproteins synthesized in plant cells

show greater similarity to the native proteins than those produced in

recombinant microorganisms. However, there are some limitations, such

as the instability of recombinant proteins in plant cell culture media and

the low protein yields reported so far for plant cells in culture (Hellwig et

al., 2004). Among therapeutic proteins, GM-CSF has been produced in

Nicotiana tabacum (Lee and Kim, 2002) and Lycopersicum esculentum

(Kwon et al., 2003). A concrete example of the application of plant cells is

the ﬁrst market license issued in January 2006 to a veterinary vaccine

produced in plant cells (Katsnelson et al., 2006). The North American

USDA (United States Department of Agriculture) considered the veter-

inary viral vaccine safe and effective in protecting chickens from New-

castle disease. This subunit vaccine consists of a protein expressed in

recombinant tobacco cells and represents the ﬁrst vaccine obtained from

plant cells to be approved worldwide.

Another possible system for the production of therapeutic proteins

consists of transgenic animals. Although there is controversy regarding

safety and reproducibility, many reports on this subject have been pub-

lished and some proteins derived from these systems are currently under

clinical trials. Parker et al. (2004) puriﬁed and characterized human

fetoprotein Æ secreted in transgenic goat milk. This protein, used in the

treatment of autoimmune diseases, proved to be identical to the native

form by mass spectrometry, circular dichroism, and pharmacokinetic tests.

Using a different approach, some authors propose post-translational

modiﬁcation in vitro. Glycosylated proteins could be obtained by in vitro

glycosylation after their production in non-mammalian expression sys-

tems. Synthetic strategies for preparing glycoproteins can be classiﬁed into

two main categories: convergent and sequential. In the convergent strat-

egy, the glycan is assembled separately by enzymatic or chemical methods,

or obtained from natural sources, and is then attached to the polypeptide

chain. In the sequential strategy, a mono- or disaccharide unit is intro-

duced into the polypeptide, and then the complete glycan is synthesized

by the sequential attachment of sugars by the action of glycosyltrans-

ferases. However, the development of simple and reliable methods for

obtaining selectively and uniformly glycosylated proteins is still a chal-

lenge and will probably remain a focus of future research (Khmelnitsky,

2004).

Finally, some authors propose the use of enzyme mixtures for the

biosynthesis of complex molecules. A cell free protein synthesis (CFPS)

system is a novel approach that was successfully used for the production

of complex mammalian proteins with multiple disulﬁde bonds (Bhatta-

charya, 2004). However, even if the technical potential of this method is

proved, its economical feasibility has still to be proven.

16.4.4 Second-generation biopharmaceuticals

Most biopharmaceuticals initially approved, known as ﬁrst-generation

biopharmaceuticals, consist of simple replacement proteins with an amino

acid sequence identical to the native human protein. Second-generation

biopharmaceuticals are engineered molecules resulting from modiﬁcations

Recombinant therapeutic proteins 405

of glycans or of amino acid composition, or from conjugation to other

molecules, such as in the case of pegylated proteins. These alterations in

the protein are carried out with different goals: (i) to improve the

pharmacokinetic proﬁle of the protein; (ii) to increase protein half-life;

and/or (iii) to alter its immunogenic proﬁle.

An example of a second-generation biopharmaceutical obtained by site-

directed mutagenesis is TNKase (Tenecteplase

), which is an altered

form of recombinant tPA produced in CHO cells by Genentech. The

substitution of three amino acids in the native protein results in a molecule

with higher afﬁnity for its receptor (ﬁbrin), a higher resistance to its

natural inhibitor (PAI-1), and an increased half-life (Walsh, 2004).

Second-generation insulins are also available and consist of molecules

with changes in one to two amino acids, aimed at accelerating the onset of

activity (Lispro and Aspart insulin products) or increasing the half-life

(Glargine insulin product).

Humanized antibodies, discussed in more detail in Chapter 17, are also

considered second-generation molecules. By humanization, the high im-

munogenicity of a murine antibody is eliminated, enabling improved

therapeutic use.

Protein engineering can also be used for creating fusion proteins or

hybrid biopharmaceuticals. An example of this type of product is Enbrel

(Etanercept), which consists of the extracellular ligand-binding domain of

the 75-kDa receptor for tumor necrosis factor-alpha (TNFÆ) and the Fc

portion of human IgG1. TNF is a proinﬂammatory cytokine present at

high concentrations in patients suffering from rheumatoid arthritis. En-

brel

acts as competitive inhibitor of TNF, as it competes with the

receptor molecules (TNFR) present in the cell. The antibody portion of

the fusion protein increases the half-life in blood (Walsh, 2004).

Another approach relies on modifying the glycan of glycoproteins to

improve pharmacokinetics of biopharmaceuticals. An example was the

introduction of two additional N-glycosylation sites in EPO, resulting in

a hyperglycosylated erythropoietin product (Aranesp

), which was ap-

proved for commercialization in 2001. The modiﬁcation of the carbo-

hydrate structure increased the half-life to 25.3 hours (as compared with

8.5 hours for the ﬁrst-generation EPO), allowing a decrease in injection

frequency that is attractive for patients.

With the considerable evolution achieved in recent years in the elucida-

tion of structure–function relationship of proteins, as well as in the areas

of protein engineering and bioinformatics, many new developments are

expected in the ﬁeld of new-generation biopharmaceuticals. These will

have signiﬁcant advantages for patients, including lower immunogenicity,

lower frequency of injections, and enhanced stability in serum.

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408 Animal Cell Technology

Monoclonal antibodies

Wirla M.S.C. Tamashiro and Elisabeth F.P. Augusto

17.1 Introduction

Antibodies are molecules secreted by plasmocytes; these are differentiated

from B lymphocytes following previous contact with a speciﬁc antigen.

The number of different antibodies produced by humans is extremely

large, incorporating between 10

and 10

distinct molecules, but any one B

lymphocyte only produces antibodies against a single speciﬁc antigen.

After immunization or contact with the antigen, the B cells which

recognize each antigenic determinant in an antigen molecule will produce

clones, thus increasing the number of lymphocytes speciﬁc for that

antigen. Although the antibodies produced by all of these lymphocyte

clones will be speciﬁc for the same antigen, these antibodies will not

necessarily be structurally identical, that is, they are polyclonal antibodies.

The result of this process is an increase in the level of speciﬁc antibodies in

the serum or body secretions of the immunized individual. Even immuni-

zation with highly puriﬁed antigens results in the production of polyclonal

antibodies, so that the composition and properties of immune sera vary

with each preparation (Abbas et al., 2000).

Attempts to produce homogeneous antibodies in vitro arose almost as

soon as lymphocytes and their biological properties were discovered.

However, B lymphocytes normally do not survive in cell cultures. For

survival, they must undergo a malign transformation, such as that induced

by the Epstein–Barr virus (Steinitz et al., 1977). Some tumors of B

lymphocytes, the multiple myelomas, can arise spontaneously and others

can be induced by the administration of mineral oil. These tumors,

whether spontaneous or induced, can be adapted to in vitro cultivation,

and they will secrete highly homogeneous antibodies (monoclonal anti-

bodies, mAbs) since they originate from a single tumor cell (Horibata and

Harris, 1977). However, even if all antibodies are homogeneous in a

myeloma culture, it is impossible to predict what this speciﬁcity will be.

The production in vitro of mAbs with a predetermined speciﬁcity has

only been possible since the advent of the technology of hybridomas,

which was introduced by Kohler and Milstein in 1975. These hybridomas

are the products of in vitro fusion of myelomas with normal B lympho-

cytes. The fusion products preserve the capacity for self-propagation in a

culture, as well as the secretion of the antibodies of interest, characteristics

inherited from the parent myeloma and the normal B lymphocyte,

respectively. The myelomas used in such fusions generally involve cell

lines from B-lymphocyte tumors developed in mice or rats (Cotton and

Milstein, 1973; Ko

hler and Milstein, 1975b; Shulman et al., 1978), while

the B lymphocytes come from mice or rats previously immunized with the

antigen of interest. After the fusion of the myeloma with the normal B

lymphocyte, the hybridomas of interest can be selected and cloned to

obtain an unlimited quantity of homogeneous antibodies that are highly

speciﬁc (Ko

hler and Milstein, 1975a).

The production of such mAbs using hybridoma technology has made it

possible to detect and quantify a wide variety of molecules produced by

live organisms in highly speciﬁc and sensitive assays, thus making possible

enormous advances in various areas of biological research. The speciﬁcity

of these mAbs has also facilitated the improvement of clinical diagnoses, as

well as creating expectations about their use as a therapeutic agent for

human diseases.

One problem with the use of these antibodies in humans, however, is

the immunogenicity (the ability to induce an immune response) of murine

mAbs, and various associated adverse reactions. Some mouse mAbs have,

however, been licensed for use in human patients (Lin et al., 2005). The

adverse reactions can be controlled to a certain extent by ‘‘humanization’’

of the murine reagents (produced by cells transfected with antibody

genes), as well as by the use of recombinant DNA techniques to produce

antibody fragments preserving the antigen-binding capacity of the original

antibody molecules (Huhalov and Chester, 2004). Only recently has the

establishment of a line of human myeloma cells (called Karpas 707H) been

reported, and this is proving useful in the production of hybridomas via

fusion with B lymphocytes obtained from immunized or infected humans

(Karpas et al., 2001; Vaisbourd et al., 2001). Obtaining all these highly

speciﬁc mAbs, whether murine, humanized, or antibody fragments, is

dependent upon the production of a hybridoma.

Both hybridomas and humanized antibody producer cells can be culti-

vated indeﬁnitely in conventional cultures, usually containing fetal bovine

serum in the medium. In the supernatants of these cultures, the quantity of

mAbs varies from 20 to 100 g/ml of protein, depending on the cell and

the system of cultivation.

There are, however, in vivo methods of obtaining mAbs, and these

produce much larger quantities. One of these is intraperitoneal administra-

tion of the hybridomas into histocompatible animals (of the same cell line

as the parents of the hybridoma) or immunodeﬁcient animals (individuals

with no functional immune system). These receptor animals will develop

ascitic tumors containing from 1 to 40 mg/ml of the mAb secreted by the

hybridoma (Kretzmer, 2002). However, despite the fact that this method

is well documented and has been used widely in the past, the procedure is

presently being rejected because of ethical concerns over the use of

laboratory animals.

This has led to an increased interest in methods for the in vitro

production of large quantities of mAbs. Submersed cultivation in bioreac-

tors is one possibility. In contrast with traditional methods, this procedure

uses large quantities of culture medium, increasing the scale of the process

and making possible the production of virtually unlimited quantities of

mAbs by a given hybridoma.

In this chapter, the fundamentals of hybridoma technology are dis-

cussed and the most widely used protocols and procedures are presented.

410 Animal Cell Technology

The main advances in large-scale in vitro production of mAbs are also

presented. This topic is of extreme importance in the biotechnology

industry, as can be seen by the extent of the use of mAbs in diagnostic and

therapeutic applications.

17.2 Antibodies

Antibodies constitute the prototype of the superfamily of immunoglobins

(Ig), molecules, which have globular domains resulting from the existence

of cysteine residues along their polypeptide chains. These residues form

disulﬁde bridges (S-S) within and between chains. The functional units of

the antibody molecules are generally monomers, each formed by a pair of

light polypeptide chains of approximately 25 kDa each, and another pair

of heavy ones of about 50 kDa (Figure 17.1). The chains of each pair are

identical, with all four chains linked by inter-chain disulﬁde bridges. Each

of the antibody chains in this monomer has an N-terminal domain,

identical for the two members of each pair, although these domains vary

from one molecule to another. The heavy chains also have three or more

constant domains (C-terminals), whereas the light chains have only a

single one. The variable N-terminal domains of the Ig molecule constitute

the antigen combining sites, with each of the two sets of heavy and light

chains of the monomer constituting a single site for antigen binding.

Hence, each monomer of the immunoglobulin has duplicate sites for

combining with a speciﬁc antigen. The rest of the molecule

(the C-domains) effects the other functions of the antibody: interaction

with the cells of the immune system (via Ig receptors), linking to the

molecules of the complement system, and crossing the placenta or epithe-

Mouse

Human

CDR1

CDR2

CDR3

CDR1

CDR2

CDR3

S-S

Figure 17.1

Structure of an antibody molecule.

Monoclonal antibodies 411

lial mucosa. The Ig classes (IgD, IgM, IgG, IgE, and IgA) are determined

by the primary sequence of the constant domains of the heavy chain pair.

IgD and IgM monomers are expressed on the surface of the naı

ve B

lymphocyte, which constitute B-cell receptors (BCRs) for antigens. Except

for IgD, all of the Ig classes can be secreted by plasmocytes upon contact

with a speciﬁc antigen, whether a foreign substance or an altered native

constituent. Although IgG and IgE are always monomers, IgA molecules

can be secreted as either monomers or dimers. IgM is always secreted as a

pentamer. In humans, there are four IgG subclasses: IgG1, IgG2, IgG3,

and IgG4, whereas there are two IgA subclasses: IgA1 and IgA2 (Abbas et

al., 2000).

There are, however, enormous numbers of antigens that may be encoun-

tered by the immune system. To deal with them, B lymphocytes undergo

a unique developmental process involving genetic recombination of gene

segments to form a wide variety of antibodies. This process, which takes

place in the bone marrow of mammals and the Fabricius bursa of birds,

was determined some 30 years ago by Tonegawa et al. (1978). The critical

aspect of this process is the occurrence of cuts in the parent DNA chain

of the developing lymphocyte by special enzymes (RAG1 and RAG2),

that are only found in these cells. This cutting and the subsequent re-

arrangement of gene segments allows great variety in the gene, which is

responsible for the formation of the N-terminal (variable) regions of the Ig

chains. This enables coupling with the wide variety of antigen determi-

nants encountered by the organism.

Three regions of the parent DNA heavy chain are affected by these cuts:

V, D, and J (for variable, diversity, and junction, respectively). Initially,

simultaneous cuts occur in a D and a J region of the chain; the segment

between the two cuts is eliminated, and the two loose ends are joined in a

DJ sequence. Then, two additional cuts occur, one in a V region, and the

second immediately prior to the new DJ sequence. The intervening

segment is eliminated, and the V region is attached to the DJ sequence to

form the ﬁnal VDJ gene sequence, which codiﬁes the variable portion of

what will be the heavy Ig chain. Figure 17.2 shows this gene recombination

in the formation of the heavy chain variable region.

The second step in the formation of the heavy chain involves transcrip-

tion of the DNA to form the primary transcript of the RNA of the

developing lymphocyte, which includes the new VDJ sequence. This

primary transcript (tRNA), however, still includes non-codifying seg-

ments (introns). In the third step, these introns are eliminated, and the

ﬁnal RNA includes only the variable N-terminal gene and the genes for all

of the constant regions.

This mRNA is then translated to heavy polypeptide chains in the

cytoplasm. The ﬁrst set formed is the heavy chain of IgM, consisting of a

VDJ region and a C region (the ﬁrst of the sequence of constant genes)

(Maki et al., 1980). Each pair of these heavy chains is joined with a pair of

surrogate light chains (formed without genetic recombination) and mi-

grates to the surface of the developing lymphocyte. This provisional

antibody probably attaches to a self-antigen, triggering the rearrangement

of the genes for the variable region of the light chains. This rearrangement

is similar to that undergone in the formation of the heavy chain, except

412 Animal Cell Technology

that only a single pair of cuts in the V and J regions is involved, because

there is no D region in light chains.

The ﬁrst or primary transcript of this light chain gene segment includes

both the VJ gene sequence and the two genes for the constant region

(kappa and lambda). The formation of the mRNA for the light chains is

similar to that of the heavy chains. Kappa chains are the ﬁrst light chains

to be formed and lambda chains will only be formed if the molecules are

non-functional.

Once the deﬁnitive light chains have been produced, they replace the

surrogate chains in the constitution of the antigen receptor of developing

B lymphocyte, and then the immature lymphocyte migrates to the periph-

eral lymphoid organs (spleen, lymph nodes, etc.). Once deﬁnitive chains

have been produced, their presence inhibits rearrangement of the genes of

the corresponding allele. This process, called allele exclusion, leads to the

production of surface immunoglobulins capable of recognizing only one

speciﬁc antigenic determinant. The set of genetic events described here is

responsible for the generation of the enormous repertoire of antibodies in

organisms with an immune system (Sakano et al., 1980).

During the mid-1970s, this process of antibody generation was not

understood, and the laboratory of Cesar Milstein was one of those trying

to decipher the mechanisms involved. The experimental approach adopted

DNA

IgM gene

IgG gene

DNA re-arrangement

V D

VV V

1 2 100

, ...

(variable)

segments

⫺

DD D

12 30

, ...

(diversity)

segments

⫺

J1, J2...J6

(joining)

segments

1234...100 1 2...30 1...6

mRNA

VDJ

Figure 17.2

Genes for the expression of immunoglobulins heavy chain.

Monoclonal antibodies 413