Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy
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ance. Since several animal cell lines can grow in serum-free media, or in
media supplemented with certain components found in bovine fetal serum,
as part of good practice for cell cultivation, the use of bovine fetal serum
should be avoided (Van Valk et al., 2004).
Within the last few years there has been a strong recommendation from
regulatory agencies such as the FDA (Food and Drug Administration,
USA), not to use animal-derived components in media formulation for the
production of biopharmaceuticals and other compounds destined for
therapeutic application in humans.
5.4 Strategies to formulate serum-free culture media
The first culture medium with a defined composition was developed in the
1950s. In 1959, Eagle developed the ‘‘minimal essential medium’’ for
mammalian cell culture, composed of glucose, amino acids, vitamins, and a
solution of balanced salts. In 1962 Grace accomplished the same with a
culture medium for insect cells (Grace, 1962). Within the last 50 years
there has been considerable work in the search for more efficient culture
media or to meet the specific needs of some cell lines. Such media were
always based on minimal formulations supplemented with fetal serum,
which as previously discussed, acts as a source of enzymes, hormones, and
microelements necessary for cell growth. However, since adding serum
has many disadvantages, the development of serum-free media with a
defined composition has intensified in recent years. Formulating medium
for animal cells requires an evaluation of the effect of each constituent so
that the optimal composition is attained.
Any new formulation may be evaluated against the properties of a
serum-substituted medium for growth and productivity. As well as
growth promotion, a serum supplement in medium may also lead to a
reduction in cell death by apoptosis and this allows extended culture
longevity. This can enable a substantial increase in recombinant protein
productivity.
In a multi-component system such as the cell environment, many factors
are interconnected, and a specific compound may be essential in some but
not all situations. Although an initial medium formulation is often based
on the composition of the circulating fluid of the organism, some nutrients
essential for metabolic activity in tissues and organs may be completely
unnecessary for in vitro cell proliferation.
One approach to developing a medium with a defined composition is to
identify the existing elements in fetal serum that have a positive effect in
cell culture. Serum may then be substituted by adding specific components
with similar functions, based on the effect of each supplement on cell
growth (Barnes and Sato, 1979, 1980; Barnes et al., 1984a, 1984b, 1984c,
1984d; Taub and Livingston, 1981). To determine the importance of a
specific component, cultures can be monitored with or without its pre-
sence at quantities normally found under physiological conditions. After-
wards, the concentrations can be varied and the effect of each specific
component can be optimized.
122 Animal Cell Technology
The key strategy when a new medium is developed is to reproduce the
metabolic conditions of the cells when in vivo. Therefore, when adding
media components it is important to maintain the pH, redox potential, and
osmolality constant, as well as attempting to avoid generation of metabolic
compounds that are toxic or growth inhibitory, such as lactate and/or
ammonium. Apart from purely nutritional factors, it is important to
develop a medium that is stable under normal storage conditions or in
contact with light.
Currently, due to the wide use of cell culture in the biopharmaceutical,
therapeutic, and diagnostic areas, the development of new media has been
critical, in an attempt to minimize the number of components, maximizing
efficiency, or simplifying the purification processes. Thus, media with
many characteristics and distinct functions have been developed.
In the development of an alternative culture medium for a given cell
line, it must be ensured that the components not being studied are
provided in sufficient quantity, such as oxygen, which can behave as a
limiting substrate in some situations. The assessment of cellular perform-
ance in different culture media is usually challenging and time-consuming,
since it requires establishment of the concentration of total and viable
cells, over multiple passages. In addition it may involve the quantification
of residual nutrients, such as glucose and glutamine, of metabolic bypro-
ducts, such as lactate, ammonium and alanine, and the concentration of the
target product. Equally relevant is the use of cellular inoculum of appro-
priate age and concentration. One of the greatest difficulties is to design a
culture medium that ensures replicate cultures that show stable genetic,
metabolic, and kinetic behavior.
Among the strategies used to establish new formulations are: univariate
analysis (where the effect of only one component is studied at a time),
analysis of the effect of a group of nutrients, and statistical design of
experiments.
The strategy of statistical design of experiments (Barros Neto et al.,
1995; Rodrigues and Iemma, 2005) appears to be a rational and efficient
way for a direct and quick determination of the effect and interaction
among the parameters analyzed (Montgomery and Runger, 1999). This
type of procedure results in the minimization of cost and time. This
strategy has proved to be adequate to qualitatively analyze the effects of
the use of protein hydrolysates as supplements for insect cell cultivation
(Ikonomou et al., 2001; Batista et al., 2005).
The new media that are commercially available possess many distinct
characteristics; some are completely free of animal-derived components,
others are serum-free, sometimes requiring addition of some protein
fraction or containing a discrete quantity of proteins. Another class is
protein-free media, which can include components derived from animals,
plants, or yeast hydrolysates. In some cases, the development of novel
media is unnecessary, as it is sufficient to supplement basal medium with a
few known specific components. It is emphasized that the final osmolality
of the formulated medium should be compatible with cellular tolerance, as
discussed in Chapter 2.
Protein hydrolysates may provide an alternative to a serum supplement.
Such hydrolysates contain oligopeptides, peptides, and amino acids ob-
Culture media for animal cells 123
tained by chemical or enzymatic hydrolysis of caseins, plants, animal
tissues, or yeast (Ikonomou et al., 2003). Some of the most commonly
used hydrolysates are bacto-peptone (Keay, 1976), proteose peptone
(Hasegawa et al., 1988), and Primatone
1
RL (Mizrahi, 1977; Schlaeger
et al., 1993; Schlaeger, 1996). Primatone
1
RL, a commercially available
meat digest, is a low-cost supplement that serves as a source of amino
acids, oligopeptides, iron salts, lipids, and traces of other low molar mass
substances.
Recently, plant hydrolysates have also been tested as additives for
serum-free culture media (Donaldson and Shuler, 1998; Franek et al.,
2000; Heidemann et al., 2000). Donaldson and Shuler (1998) demonstrated
that a peptide obtained from soy hydrolysate (Hy-Soy
1
) played an
important role in a serum-free medium developed for insect cells. Ikono-
mou et al. (2003) also showed the effectiveness of culture medium
supplemented with the soy hydrolysate SE50MAF in the growth of High
Five
TM
cells.
Further than providing nutrients, these hydrolysates may play an
essential role in providing growth factors, antiapoptotic factors, or protein
production stimulators in serum-free cultures (Ikonomou et al., 2003).
As well as these hydrolysates, other supplements can also totally or
partially substitute serum, such as yeast extract (Drews et al., 1995; Wu
and Lee, 1998; Wu et al., 1998; Ikonomou et al., 2001, 2003; Sung et al.,
2004; Batista et al., 2005, Mendonc¸a et al., 2007) and peptides obtained
from insect hemolymph (Maranga et al., 2003; Souza et al., 2005). While
yeast extract is mainly used as a source of vitamins (Mitsuhashi, 1989),
stimulating cell growth, the peptides obtained from insect hemolymph
have a potent effect in inhibiting apoptosis and positive activity in cell
growth and in producing recombinant proteins (Maranga et al., 2003).
Some cell lines, such as Namalwa, can grow satisfactorily in medium in
which the only protein is albumin. Other cell lines show distinct protein
requirements, such as albumin, transferrin, and insulin, or the addition of
polypeptide growth factors, isolated from non-serum sources that have
shown stimulation of many cell types in culture. Some cells have very
fastidious growth requirements and their stability and productivity may
be reduced significantly in serum-free media.
The chemically defined media, that is, those that may contain a specific
protein or a specific animal extract compound, as long as they are purified
and have known concentration of all components, are ideal to eliminate
production variability, increasing reproducibility, facilitating and reducing
the costs of protein purification, and eliminating the potential risk of
pathogenic agent transmission. Many components of animal origin that
are necessary for cell growth can be substituted by bioactive synthetic
chemical products or by compounds originating from microbial fermenta-
tion. The formulation of chemically defined media can allow more in-
depth studies, especially metabolic and regulation studies, as well as the
fact that their use can facilitate approval of products derived from animal
cells by the regulatory agencies.
Despite all the development seen, up to the present time there is no
standard culture medium that could be universally utilized for all cells,
since each cell line has a specific metabolic profile and requires an
124 Animal Cell Technology
appropriate medium composition. The most difficult part of the medium
development process is to predict exactly which supplement or set of
supplements will be necessary for each cell line. Nevertheless, useful
information can be obtained from work on cell lines of the same origin.
Recent advances in cellular biology and molecular genetics have allowed
the development of new cell lines and this has intensified the demand for
new culture media.
Generally, in terms of the growth factor requirements, cells can be
classified in four categories, as discussed below (Jenkins, 1991). In the first
category are the transformed cell lineages, such as CHO, BHK, melanoma,
hybridoma, and myeloma cells, which go through the cell cycle, only
needing some maintenance factors. Normally, these cells need insulin
(5 g/ml), serum albumin, lipid- and vitamin-carrying proteins, transferrin
(5 g/ml for iron transport), and microelements (Jenkins, 1991).
The second category includes mesenchymal cells, such as fibroblasts
(BALB/c 3T3, Swiss 3T3), adipocytes, endothelial cells, smooth thin
muscle cells, and neuroectodermic cells (such as glia cells). Most of these
cells need maintenance factors. Some cells, such as the NIH-3T3, can grow
in a serum-free medium containing minimal medium supplemented with
transferrin (25 g/ml), insulin (10 g/ml), EGF (100 ng/ml), bFGF (100
ng/ml), and PDGF (0.5 U/ml).
The cells from the third category, such as epithelial cells isolated from
mammal glands, testes, ovaries, pituitary gland, prostate, and from ocular
tissue, generally proliferate with more difficulty in a defined medium.
Besides maintenance factors, these cells may also need proteins from the
ECM to promote their adhesion. Those proteins can be collagen, fibronec-
tin, or laminin. Non-peptide factors, such as retinol (50 ng/ml) and
corticosteroids (10–100 ng/ml), may also be necessary.
Finally, in the fourth and last category are certain types of cells (PC12,
hybridomas, and hepatic cells) that may also need factors such as NGF,
IL-6, and growth hormones.
When preparing a new medium the need for cell adaptation should be
considered. The adjustment process for low protein media is often carried
out in static cultures employing tissue culture flasks. However, problems
seen at the time of adaptation can be minimized in suspension cultures,
since this allows subculture during the log growth phase. Cultures in
suspension also allow better medium oxygenation. However, under any
condition and with any medium, there will always be a need for some cell
adaptation to the new medium to stabilize cellular replication. As soon as
a cell population is adapted for growth in a low protein medium, it is
recommended that a cell bank is established.
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128 Animal Cell Technology
6
Post-translational
modification of
recombinant proteins
Michael Butler
6.1 Introduction
Many proteins require post-translational modifications to insure the full
biological activity that is essential for their use as efficacious biopharma-
ceuticals. The most widely recognized form of post-translational modifica-
tion is glycosylation that requires an extensive sequence of processing and
trimming events in the endoplasmic reticulum (ER) and Golgi apparatus.
Because eukaryotic cells have the metabolic capability for these types of
modifications they have become the host cells used in biopharmaceutical
processes. More specifically, mammalian cells (particularly the hamster
BHK and CHO cells) have become the host cells of choice because the
glycosylation patterns generated from these cells are most similar to
equivalent human patterns. Most therapeutic proteins are glycoproteins
that have an extensive heterogeneity of glycoforms arising from incom-
plete glycosylation processes.
Although glycosylation is the most extensive and widely studied form
of post-translational modification, there are other modifications that are
equally important for the activity of specific proteins. The blood clotting
factor, factor X, is a good example to illustrate a range of post-translational
modifications on a single complex protein. The recognized modifications
necessary to ensure bioactivity of this factor include: removal of signal
sequences, formation of disulfide bonds, gamma-carboxylation of glutamic
acid residues, modification of aspartate to beta-hydroxyaspartate, N- and
O-glycosylation, removal of an internal tripeptide, and removal of a
propeptide (Camire et al., 2000). In order to express this protein as a
recombinant form in a cell culture bioprocess, it is necessary to insure that
the host cell has the appropriate metabolic capabilities for efficient post-
translational modification and also to insure that the culture conditions are
suitable to drive the metabolism to high productivity of the active protein.
This chapter describes some of the most important and well-studied
forms of post-translational modifications of proteins, which have been
associated with the production of glycoproteins as biopharmaceuticals
from mammalian cell culture systems. Most attention is given to glycosy-
lation because of its general importance for the activity of all these
proteins. However, other modifications are also described and related to
the production of specific proteins.
6.2 Glycan structures attached to proteins
Carbohydrate chains when attached to other molecules are known as
glycans. In proteins the glycans can be attached to asparagine residues
forming N-glycans or to serine or threonine residues forming O-glycans.
6.2.1 N-glycans
This is the most widely studied structural form of glycosylation and has
the greatest effect on overall protein structure and function (Kornfeld and
Kornfeld, 1985; Spellman, 1990). The glycan consists of a core pentasac-
charide (Man
3
GlcNAc
2
) attached via an N-glycosidic bond to an Asn
residue of a consensus amino acid sequence (sequon) Asn-X-Ser/Thr,
where X can be any amino acid except proline (Figure 6.1).
The full sequence of reactions involved in the synthesis of the N-glycans
in mammalian cells is shown in Figure 6.2. The precursor for glycosylation
is the oligosaccharide (Glc
3
Man
9
GlcNAc
2
), which is formed by the step-
wise addition of monosaccharides from nucleotide sugars (UDP-GlcNAc
and GDP-Man) or lipid intermediates (Dol-P-Man and Dol-P-Glc)
to a dolichol-linked pyrophosphate acceptor to form the complex:
Glc
3
Man
9
GlcNAc
2
-P-P-dolichol. The 14-mer oligosaccharide is trans-
ferred to the consensus sequence (Asn-X-Ser/Thr) on the growing peptide
via the complex reaction of the oligosaccharyltransferase (OST) enzyme,
which is thought to be approximately 65 amino acid residues from the
ribosome P-site (Yan and Lennarz, 2005). Further processing by trimming
of the attached glycan occurs by glucosidases and mannosidases that are
Asn
Mannose
GlcNAc
Sequon Asn-X-Ser/Thr⫽
Figure 6.1
Core N-glycan structure. This consists of a core pentasaccharide composed of a
trimannosyl group attached to two Glc-NAc residues. The structure is attached to
an asparagine (Asn) residue in a sequon which is an amino acid motif of three
amino acids. X is any amino acid (except Pro) that is positioned between Asn and
either serine or threonine.
130 Animal Cell Technology
located within the membrane of the ER. Trimming is initiated by Æ-1,2
glucosidase I (Gluc I) which removes the terminal glucose. The next two
glucose residues are then removed sequentially by a single Æ-1,3 glucosi-
dase II (Gluc II). At least one mannose residue may then be removed by
mannosidase I (Man I) before the newly synthesized glycoprotein is
transported to the Golgi cisternae by means of vesicles. This sequential
process of trimming in the ER will only occur efficiently if the protein
assumes an appropriate folding pattern and so the process has been
thought of as a quality control mechanism required for the formation of
the protein tertiary structure (Ellgaard and Helenius, 2003).
The core structure may be processed further to a complex-type glycan
with the sequential addition of monosaccharides such as GlcNAc, galac-
tose, fucose, and sialic acid through a series of transferases that are present
in the Golgi. In mammalian cells high-mannose glycan structures are
produced as intermediates in the formation of complex glycans (Figure
6.3). These may have two to six additional mannose structures attached to
the core as a result of processing in the ER prior to translocation to the
Asn Xaa Ser/Thr
Dol
P
P
NH
2
Golgi
Hybrid
type
Man
Complex
type
Oligosaccharide
transferase
Glc
Glc
Glc
Endoplasmic
reticulum
α-Glc I α-Glc II
α-Glc II α-Man I
Man
Oligomannose
type
GnTI
SialT
FucT
GalT
GnTII
Man II
Figure 6.2
N-linked glycosylation pathway. The addition of a 14mer oligosaccharide to an N-glycan sequon
occurs co-translationally in the endoplasmic reticulum. This is followed by a series o f trimming
reactions to remove glucose and mannose residues. In animal cells the glycan structures with
exposed mannose groups and attached to newly synthesised proteins are transferred to t he Golgi for
further processing. A series of transferase enzymes ensures the addition of N-acetylglucosamine,
fucose, galactose, and sialic acids. The transferase reactions form a metabolic network resulting in a
variety of end products, which account for the variable glycoforms found in pr oteins.
Post-translational modification of recombinant proteins 131