Glycosylation and Its importance in animal cell cultures

Glycosylation and animal cells in culture

The characterization of the critical parameters of glycosylation
should enable process control to reduce the heterogeneity of
glycoforms so that production processes are consistent.

Further improvement may also be made by the identification of

glycoforms with enhanced biological activity to enhance clinical
efficacy.
For the production of a recombinant protein as a
biotherapeutic it is essential to ensure that a
consistent glycosylation profile is maintained
between batches (Restelli and Butler 2002).

However, this may not be so easy to control

given that the extent of glycosylation may
decrease over time in a batch culture (Curling et
al. 1990).

This is likely to be due to the depletion of

nutrients, particularly glucose or glutamine,
which have been shown to limit the glycosylation
process (Hayter et al. 1992; Nyberg et al. 1999).
Fedbatch strategies should also be designed to ensure that the
concentrations of these key nutrients do not decrease to a
critical level that could compromise protein glycosylation (Xie
andWang 1997).

These lower levels were found to be <0.1 mM glutamine and

<0.7 mM glucose for the production of γ-interferon from CHO
cells (Chee et al. 2005).
Non-optimal pH conditions (<6.9 and >8.2) have also been shown to
alter the pattern of glycosylation (Rothman et al. 1989; Borys et al.
1993).

Reduced terminal galactosylation has been shown in the glycans of

immunoglobulin (IgG) produced under low oxygen conditions (Kunkel
et al. 1998).

Nabi and Dennis(1998) observed an increase in the polylactosamine

content of a protein produced at lower temperatures and attributed this
to changes in the transit time through the Golgi.
The pattern of protein glycosylation is dependent on the
expression of various glycosyltransferase enzymes that are
present in the Golgi of the cell.

Differences in the relative activity of these enzymes among

species can account for significant variations in structure.

In one systematic study of glycan structures of IgG produced

from cells of 13 different species, significant variation was
found in the proportion of terminal galactose, core fucose and
bisecting GlcNAc (N-acetylglucosamine )(Raju et al. 2000).

The structure of sialic acid may also vary, with N-glycolyl-

neuraminic acid (NGNA) found in goat, sheep and cows rather
than the N-acetylneuraminic acid (NANA) found in humans.

NGNA is the predominant sialic acid in mice, but CHO-produced

glycoproteins have predominantly NANA, although a small
proportion (up to 15%) of NGNA can occur (Baker et al. 2001).

These differences in glycan structure are important tential

immunogenicity of these structures in humans.
The production of specific protein glycoforms may allow the possibility
of even more efficacious drugs (Shriver et al. 2004). Functional
glycomics is an expanding area of science that attempts to understand
the physiological function of specific carbohydrate groups.

This approach established the importance of the sialylation of EPO

with the discovery that the removal of sialic acid groups from the
glycans resulted in a significantly reduced half-life in the blood stream
(Erbayraktar et al. 2003).

Protein engineering has allowed the creation of a modified EPO with

two extra glycan attachment sites and with the potential to
incorporate eight extra sialic acid groups per molecule. This has led to
a new generation EPO called darbepoetin , which has a three times
higher drug half-life (Egrie et al. 2003).

This strategy of enhancing the half-life of a biotherapeutic has also

been successful for other recombinant proteins such as follicle
stimulating hormone (Perlman et al. 2003) and thyroid stimulating
hormone (Thotakura et al. 1991).
Complete glycosylation of recombinant proteins is usually associated
with maximisation of galactosylation and sialylation. Often these two
processes are incomplete and this gives rise to considerable glycan
structural variation.

CHO cells can be engineered with a combination of human β1,4-

galactosyltransferase and α2,3-sialyltransferase to ensure high
activities of these enzymes.

The recombinant proteins produced by these cells exhibited greater

homogeneity compared to controls and increased terminal sialic acid
residues (Weikert et al. 1999).

An alternative approach involves glycoengineering of the proteins in

vitro (Raju et al. 2001).

Preparations of these terminal transferase enzymes can be immobilized

so that glycoproteins can be galactosylated and sialylated in the
presence of appropriate galactose and sialic acid donors.
Structural changes of glycans can also be brought about
by metabolic engineering of the host cell line.

This includes gene knockout of already expressed

glycosyltransferases or the insertion of novel activities (Weikert
et al. 1999).

The presence of a bisecting N-acetylglucosamine (Umana et al.

1999; Davies et al. 2001) or the absence of fucose (Shields et
al. 2002; Shinkawa et al. 2003; Okazaki et al. 2004) in the
conserved glycan of an IgG antibody has been shown to
enhance attachment to Fc receptors and result in an increase in
antibody-dependent, cell-mediated cytotoxicity (ADCC). This has
been of value in the design of antibody therapeutics.
Recent work with Herceptin, which is a novel
humanized antibody approved for the treatment of
breast cancer, has shown that a glycoform with no
fucose has a 53 times higher binding capacity to an Fc
receptor that triggers its therapeutic activity (Shinkawa
et al. 2003).

This enhancement of antibody-dependent, cell-

mediated cytotoxicity (ADCC) allows the antibody to be
effective at lower doses.

Afucosylated antibodies can be produced from cells in

which the gene for fucosyl transferase has been
removed by gene knockout technology.
NUTRITIONAL REQUIREMENTS FOR
ANIMAL CELL CULTURE

Basic Constituents of
media

Inorganic salts
Carbohydrates
Amino Acids
Vitamins
Fatty acids and lipids
Proteins and peptides
Serum
The term complete medium implies a medium with all its
constituents and supplements added and sufficient for use
specified.

Defined media range in complexity from relatively simple Eagle’s

MEM which contains essential amino acids, vitamins and salts, to
complex media such as M119, RPMI and F12 and wide range of
serum free formulations(MCDB-CHO cells, Iscove’s-Lymhoid cells).

Complex media contain larger number of nonessential amino

acids, extra metabolites like nucleosides, lipids, TCA cycle
intermediates and minerals.
Inorganic Salts

The inclusion of inorganic salts in media performs several

functions. Primarily they help to retain the osmotic balance of
the cells and help regulate membrane potential by provision of
sodium, potassium and calcium ions.

All of these are required in the cell matrix for cell attachment
and as enzyme cofactors.

Divalent cations, particularly Ca2+

1. are required by some cell adhesion molecules, such as
cadherins.
2.important signal transduction intermediate.
3.Can influence whether cells will proliferate or differentiate.

SO42-, PO43- and HCO3- have roles as anions required by the

matrix and nutritional precursors for macromolecules, as well
as regulators of intracellular charge.

Sodium bicarbonate conc. is determined by the conc. of

CO2 in gas phase and has significant nutritional role.   
The role of the calcium-sensing receptor in epidermal differentiation
Calcium regulates the proliferation and differentiation of
keratinocytes both in vivo and in vitro. Elevated
extracellular Ca2+ concentration ([Ca2+]o) raises the
intracellular free calcium ([Ca2+]i) and activates
differentiation-related genes. Cells lacking the calcium-
sensing receptor (CaR) fail to respond to [Ca2+]o and to
differentiate, indicating a role for CaR in keratinocyte
differentiation. These concepts derived from in vitro
experiments have been tested and confirmed in two mouse
models.
Osmolarity of the culture medium is very important
since it regulates the flow of substances in and out
of the cell.Salts are the major components
contributing to the osmolarity of the medium.

Osmolarity is controlled by the addition or

subtraction of salt in the culture. All commercial media
are formulated in such a way that their final osmolarity is
around 300 mOsm.

Evaporation of culture media from open culture will

rapidly increase osmolarity resulting in stressed or
damaged cells.

Open culture systems should have incubators with

high humidity levels to reduce evaporation.
Carbohydrates

The main source of energy is derived from carbohydrates generally in the

form of sugars. The major sugars used are glucose and galactose however
some media contain maltose or fructose.

The concentration of sugar varies from basal media containing

1g/l(Eagle’s minimum essential medium) to 4.5g/l(Dulbecco’s modified
medium-DMEM) in some more complex media. Media containing the
higher concentration of sugars are able to support the growth of a wider
range of cell types.

The accumulation of lactic acid in the medium particularly evident in

embryonic and transformed cells, implies that TCA cycle may not function
entirely as it does in vivo, and recent data has shown that much of the
carbon source is derived from glutamine rather than glucose.

This finding explains the exceptionally high requirement of some cultured

cells for glutamine or glutamate.
  Buffering Systems

Most cells require pH conditions in the range 7.2 - 7.4. There are major
variations to this optimum.

Fibroblasts prefer a higher pH (7.4 - 7.7) whereas, continuous

transformed cell lines require more acid conditions pH (7.0 - 7.4).

The optimum pH is essential to maintain the proper ion balance,

optimal functioning of cellular enzymes and binding of hormones and
growth factors to cell surface receptors in the cell cultures.

Regulation of pH is particularly important immediately following cell

seeding when a new culture is establishing and is usually achieved by
one of two buffering systems
BUFFERING SYSTEMS

(i) A "natural" buffering system where gaseous CO 2

balances with the CO3 / HCO3 content of the culture medium
.

(ii) Chemical buffering :Good et al used a range of

Zwitterionic buffers like N-2 hydroxyethyl piperazine N’-2
ethane sulfonic acid (HEPES) having pKa 7.31 optimal for
cell culture.

HEPES has superior buffering capacity in the pH range 7.2 -

7.4 but is relatively expensive and can be toxic to some cell
types at higher concentrations.

HEPES buffered cultures do not require a controlled

gaseous atmosphere. These buffers do not penetrate cell
membrane and equilibrate with air.
 Buffering
Cultures using natural bicarbonate/CO2 buffering systems
need to be maintained in an atmosphere of 2%-10% CO 2 in
air usually supplied in a CO2 incubator.

Incubators are used routinely to provide the correct growth

conditions, such as temperature, degree of humidity and CO 2
levels in a controlled and stable manner.

The inclusion of pyruvate in the medium enables cells to

increase their endogenous production of CO 2, making them
independent of exogenous CO2, as well as HCO3-

Most commercial culture media include phenol red as a pH

indicator so that the pH status of the medium is constantly
indicated by the color.

Usually the culture medium should be changed / replenished

if the color turns yellow (acid) or purple (alkali).
 Temperature
Apart from its direct effect on cell growth, the temperature
influences pH due to the increased solubility of CO2 at lower
temperature and possibly because of changes in ionization and
pKa of buffer.
pH should be adjusted to 0.2 units lower at room temperature
than at 370C.
AMINO ACIDS
Amino acids, referred to as the essential amino acids, cannot be synthesized
by adult vertebrate animals and thus must be obtained from their diet.

Animal cells grown in culture also must be supplied with these amino acids,
The 12 essential amino acids are: L-arginine; L-cystine; L-glutamine; L-
histidine; L-isoleucine; L-leucine; L-methionine; L-phenylalanine; L-
threonine; L-tryptophan; L-tyrosine; and L-valine.

In the intact animal, cysteine, glutamine, and tyrosine., are synthesized by

specialized cells; for example, liver cells make tyrosine from phenylalanine,
and both liver and kidney cells can make glutamine.

Animal cells in culture can synthesize the 8 remaining amino acids; thus
these amino acids need not be present in the diet or culture medium.

The other essential components of a medium for culturing animal cells are
vitamins, which the cells cannot make at all or in adequate amounts; various
salts; glucose; and serum, the noncellular part of the blood
Part of glutamine is deaminated to yield ammonia and glutamate which
is converted to other amino acids for biosynthesis purposes.

Glutamine also enters into the TCA cycle to yield carbon skeletons for
other aminoacids and to yield ATP, CO2 and H2O.
 Proteins and Peptides

These are particularly important in serum free media. The most

common proteins and peptides include albumin, transferrin,
fibronectin and fetuin and are used to replace those normally
present through the addition of serum to the medium.

Fetuins are blood proteins, which are made in the liver and
secreted into the blood stream.

They belong to a large group of binding proteins mediating the

transport and availability of a wide variety of cargo substances
in the blood stream.

The best known representative of these carrier proteins is

serum albumin, the most abundant protein in the blood plasma
of adult animals.
Fetuin is more abundant in fetal blood, hence the name fetuin
(from lat. fetus). Fetal calf serum contains more fetuin than
albumin, while adult serum contains more albumin than
fetuin.

Proteins increase the viscosity of the medium, reducing shear

stress during pipetting and stirring and may add to the
medium’s buffering capacity.
Fatty Acids and Lipids
Like proteins and peptides these are important in serum free
media since they are normally present in serum. e.g. cholesterol
and steroids essential for specialized cells, cholesterol helps in
membrane synthesis.

Trace Elements
These include trace elements such as zinc, copper, selenium and
tricarboxylic acid intermediates. Selenium is a detoxifier and helps
remove oxygen free radicals. They also are enzyme cofactors
.
Vitamins
Serum is an important source of vitamins in cell culture. However,
many media are also enriched with vitamins making them
consistently more suitable for a wider range of cell lines. Vitamins
are precursors for numerous co-factors. Many vitamins especially B
group vitamins are necessary for cell growth and proliferation and
for some lines the presence of B12 is essential. Some media also
have increased levels of vitamins A and E. The vitamins commonly
used in media include riboflavin, thiamine and biotin.
SERUM-CONTAINING MEDIUM (EAGLE'S MEDIUM)

Essential The essential amino acids — histidine,

amino acids isoleucine, leucine, lysine, methionine,
phenylalanine, threonine, tryptophan, and
valine — plus cysteine, glutamine, and
tyrosine (all at 10−4 to 10−5 M)
Vitamins Choline, folic acid, nicotinamide,
pantothenate, pyridoxal, and thiamine (all
at 1 mg/L); inositol (2 mg/L); riboflavin
(0.1 mg/L)
Salts Na+, K+, Ca2+, Mg2+, Cl−, PO43−, HCO3−

Glucose 0.9 g/L

Dialyzed 5 – 10% of total volume

serum
Advantages of Serum
The supplementation of mammalian cell culture media with
sera of animal origin remains still standard, providing
necessary nutrition, attachment factors, shear protection,
growth factors and cytokines . Of the various biological
fluids used as culture medium, serum is the most widely
used

Serum increases the buffering capacity of cultures ,that can be

important for slow growing cells or where the seeding density
is low (e.g. cell cloning experiments).

It also helps to protect against mechanical damage which may

occur in stirred cultures or whilst using a cell scraper.

A further advantage of serum is the wide range cell types with

which it can be used despite the varying requirements of
different cultures in terms of growth factors.

In addition serum is able to bind and neutralize toxins

Advantages of Serum

Serum contains insulin, a hormone required for growth of many

cultured vertebrate cells, and transferrin, an iron-transporting protein
essential for incorporation of iron by cells in culture.

Serum can reduce oxidative injury to cells caused by ferrous ions.

Heat inactivated serum can be used (incubation at 560C for 30 minutes)

to remove toxic compounds or other agents that interfere with tissue
typing assays.

A major role of serum is to supply proteins, e.g., fibronectin, which

promote attachment of cells to the substrate. It also provides spreading
factors that help the cells to spread out before they can begin to divide.
Growth hormone may be present in serum, particularly fetal serum
and in conjunction with somatomedins (IGFs) may have mitogenic
effect.

Hydrocortisone present particularly fetal bovine serum in varying

amounts can promote cell attachment and cell proliferation but
under certain conditions like high cell density, may be cytostatic and
can induce cell differentiation.
Various growth factors are present in serum at very low concentrations: for
example, growth hormone (34 ng/ml) and insulin (0.2 ng/ml).

Serum from fetal calves is used frequently because it contains higher

concentrations of certain growth factors than serum from other species.

SOURCE: H. Eagle, 1959, Science 130:432; S. E. Hutchings and G. H. Sato, 1978, Proc.
Nat'l., Acad. Sci. USA 75:901.
Beneficial effects of serum supplementation limited by several
disadvantages
Serum-free media allows users to standardize their cell culture conditions
by avoiding the use of undefined and highly variable serum products
derived from humans or animals, e.g. human AB serum or fetal calf serum
(FCS).

The high variability in the biological properties of different serum batches

makes it necessary to pre-screen many batches in order to obtain a single
one which is well suited for a given application.

For example, even a brief exposure of PBMC (peripheral blood mononuclear

cells) to a mitogenic serum batch during washing or freezing of these cells
will result in a high background in cytokine assays, and toxic/inhibitory
serum batches will jeopardize the assay results. 

Switching to, or using a different batch of serum introduces each time a

significant unpredictable variable to the test conditions, making test results
difficult to compare. 

For all these reasons, there is considerable pressure from regulatory

agencies and from the scientific community to avoid the use of serum and
move to consistent, defined substitutes. 
Media
Examples Uses
type

PBS,
Hanks BSS,
Balanced
Earles salts
salt Form the basis of many complex media
DPBS
solutions
HBSS
EBSS

Basal
MEM Primary and diploid cultures.
media

Modification of MEM containing increased level of amino

  DMEM acids and vitamins. Supports a wide range of cell types
including hybridomas.

  GMEM Glasgows modified MEM was defined for BHK-21 cells

 Originally derived for human leukaemic cells.
Complex RPMI 1640
It supports a wide range of mammalian cells
media
including hybridomas

Further enriched modification of DMEM which

  Iscoves DMEM
supports high density growth

Leibovitz L-15 Designed for CO2 free environments

 

TC 100
Grace's Insect Medium
  Designed for culturing insect cells
Schneider's Insect
Medium
Serum
Free For use in serum free applications.
CHO
Media

NOTE: These media must be supplemented with

Ham F10 and
other factors such as insulin, transferrin and
  derivatives
epidermal growth factor. These media are usually
Ham F12
HEPES buffered
DMEM/F12

Insect
Sf-900 II SFM, Specifically designed for use with Sf9 insect cells
cells
SF Insect-Medium-2
 Disadvantages of serum

Physiological Variability
Shelf life and consistency
Quality control-
Specificity
Availability
Downstream Processing-can be a major obstacle to product
purification and may even limit pharmaceutical acceptance of
the product
Contamination
Cost
Standardization
Disadvantages of serum
Growth inhibitors-Hydrocortisone present at around 1 x10-8
M in fetal serum can be cytostatic to many cell types, such as
glial cells and lung epithelium, at high cell densities and TGF-,
released from platelets is cytostatic to many epithelial
cells.Hence the net effect of the serum is the unpredictable
combination of both inhibitors and stimulation of growth.

Serum may contain some cytotoxic or potentially

cytotoxic constituents. For example, foetal calf serum
contains the enzyme polyamine oxidase, which converts
polyamines like spermidine and spermine (secreted by
fast growing cells) into cytotoxic polyaminoaldehyde.
DEFINED (SERUM-FREE) MEDIUM
Amino acids As before plus alanine and asparagine
(10−4 M)
Vitamins, salts, As before
glucose

Other additions:
   Fatty acids Linoleic acid, lipoic acid
  Nitrogen Hypoxanthine, thymidine, putrescine
compounds

Carbon source Pyruvate and glucose (0.9 g/L)

Trace elements Cadmium (Cd), manganese (Mn),
molybdenum (Mo), nickel (Ni), tin (Sn),
vanadium (V)
Hormones and Insulin, transferrin, hydrocortisone,
growth factors fibroblast growth factor, epidermal
growth factor
Serum Free Media -

In view of the disadvantages due to serum, extensive

investigations have been made to develop serum-free
formulations of culture media. These efforts were mainly based
on the following three approaches:

(1) analytical approach based on the analysis of serum

constituents,

(2) synthetic approach to supplement basal media by various

combinations of growth factors, and

(3) limiting factor approach consisting of lowering the serum

level in the medium till growth stops and then supplementing
the medium with vitamins, amino acids, hormones, etc. till
growth resumes.
In some cases metabolic analysis may help in media design. For example, NS0
myeloma cells lack a functional pathway for cholesterol synthesis and so
cholesterol is required as a lipoprotein supplement in the medium (Gorfien et al.
2000).

Protein hydrolysates from non-animal sources have been found to provide good
growth promotion in some culture systems (Sung et al. 2004).

Analysis of the depletion of media components may lead to the identification of

specific nutrients that may be required at higher supplement levels or for
inclusion in feeding regimes.

Another original approach is the identification by microarray analysis of specific

receptors expressed during cell growth, so that corresponding ligands
may be incorporated into the medium (Donahue 2004).
Advantages of Serum Free Media -

1. Improved reproducibility of results from different laboratories and

over time since variation due to batch change of serum is avoided.

2. Easier downstream processing of products from cultured cells.

3. Toxic effects of serum are avoided

. 4. Biassays are free from interference due to serum proteins.

5. There is no danger of degradation of sensitive proteins by serum

proteases.

6. They permit selective culture of differentiated and producing cell

types from the heterogenous cultures.
Some supplements such as suramin (Zanghi et al. 2000) or insulin
growth factor (Sunstrom et al. 2000) may provide independent
anti-apoptotic protection in serum-free cultures. There are also
other specific caspase inhibitors available to suppress apoptosis
(Tinto et al. 2002) but their expense in large-scale cultures is likely
to be prohibitive.

Protein Free Media -

Protein free media do not contain any protein; they only
contain non-protein constituents necessary for culture of the
cells. The formulations MEM, DME, RPM-1640, etc. are
protein free; where required, protein supplementation is
provided.
Disadvantages of Serum Free Media
1. Most serum free media are specific to one cell type. Therefore, different
media may be required for different cell lines. It turns out that producer cell lines
are quite fastidious in their growth requirements and that such requirements
vary considerably from one cell line to another.

Therefore, it has not been possible to design a single serumfree formulation to act as a
serum substitute suitable for the growth of all cell lines. In fact even different clones of
CHO cells may require different formulations for optimal growth. This has given rise to a
strong drive for the development of serum-free and animal-component-free
formulations that are tailored to the needs of specific producer cell lines.

2. Reliable serum free preparations, for most of the media formulations are
not available commercially. This necessitates time consuming task of
preparing the desired formulations in the laboratory.

3. A greater control of pH, temperature, etc. is necessary as compared to

that with serum containing media.
4. Growth rate and the maximum cell density attained are
lower than those with serum containing media.

5. Cells tend to become fragile during prolonged agitated

cultures unless biopolymers or synthetic polymers are added.

Several defined media have been evolved from the Eagle's

minimal essential medium (MEM), e.g., Dulbecco's enriched
modification (DME), Ham's F12, :,pMRL1O66, RPMIl640,
McCoy's 5A and Iscove's modified Dulbecco’s' (IMDM); all are
commercially available.

Often a 1: 1 mixture of DME and F12 is used as a serum free

formulation. If needed, purified proteins and/or hormones may
be added to the medium.