Glycosylation and Its importance in animal cell cultures

Glycosylation and animal cells in cultureThe 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 theconcentrations 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 aboutby 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.

Basic Constituents of media

Inorganic salts Carbohydrates Amino Acids Vitamins Fatty acids and lipids Proteins and peptides Serum

NUTRITIONAL REQUIREMENTS FOR ANIMAL CELL CULTURE

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-, PO4

3- 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.   

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.

The role of the calcium-sensing receptor in epidermal differentiation

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 CO2 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.

Cultures using natural bicarbonate/CO2 buffering systems need to be maintained in an atmosphere of 2%-10% CO2 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 CO2 levels in a controlled and stable manner.

The inclusion of pyruvate in the medium enables cells to increase their endogenous production of CO2, 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).

Buffering

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 ACIDSAmino 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.

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.

Proteins and Peptides

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 LipidsLike 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 ElementsThese 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.

VitaminsSerum 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 amino acids

The essential amino acids — histidine, 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 serum

5 – 10% of total volume

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

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.

Serum-free mediaSerum-free media allows users to standardize their cell culture conditions allows users to standardize their cell culture conditions by avoiding the use of undefined and highly variable serum products 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 derived from humans or animals, e.g. human AB serum or fetal calf serum (FCS). (FCS). The The high variabilityhigh variability in the biological properties of different serum batches in the biological properties of different serum batches makes it necessary to pre-screen many batches in order to obtain a single makes it necessary to pre-screen many batches in order to obtain a single one which is well suited for a given application.one which is well suited for a given application.

For example, even a brief exposure of PBMC (peripheral blood For example, even a brief exposure of PBMC (peripheral blood mononuclear cells) to a mononuclear cells) to a mitogenic mitogenic serum batch during washing or freezing serum batch during washing or freezing of these cells will result in a high background in cytokine assays, and of these cells will result in a high background in cytokine assays, and toxic/inhibitory serum batches will jeopardize the assay results. toxic/inhibitory serum batches will jeopardize the assay results. 

Switching to, or using a different batch of serum introduces each time a Switching to, or using a different batch of serum introduces each time a significant unpredictable variable to the test conditions, making test significant unpredictable variable to the test conditions, making test results difficult to compare.  results difficult to compare. 

For all these reasons, there is considerable pressure from regulatory For all these reasons, there is considerable pressure from regulatory agencies and from the scientific community to avoid the use of serum and agencies and from the scientific community to avoid the use of serum and move to consistent, defined substitutes.  move to consistent, defined substitutes. 

Beneficial effects of serum supplementation limited by several disadvantages

Media type

Examples Uses

Balanced salt solutions

PBS,Hanks BSS, Earles saltsDPBSHBSS EBSS

Form the basis of many complex media

Basal media

MEM Primary and diploid cultures.

  DMEMModification of MEM containing increased level of amino acids and vitamins. Supports a wide range of cell types including hybridomas.

  GMEM Glasgows modified MEM was defined for BHK-21 cells

Complex media

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

  Iscoves DMEMFurther enriched modification of DMEM which supports high density growth

 Leibovitz L-15 Designed for CO2 free environments

 

TC 100Grace's Insect Medium

Schneider's Insect Medium

Designed for culturing insect cells

Serum Free Media

CHOFor use in serum free applications.

 Ham F10 and derivativesHam F12 DMEM/F12

NOTE: These media must be supplemented with other factors such as insulin, transferrin and epidermal growth factor. These media are usually HEPES buffered

Insect cells

Sf-900 II SFM, SF Insect-Medium-2

Specifically designed for use with Sf9 insect cells

Disadvantages of serum

Physiological VariabilityShelf life and consistencyQuality control-SpecificityAvailabilityDownstream Processing-can be a major obstacle to

product purification and may even limit pharmaceutical acceptance of the product

ContaminationCostStandardization

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.

Disadvantages of serum

DEFINED (SERUM-FREE) MEDIUM

Amino acids As before plus alanine and asparagine (10−4 M)

Vitamins, salts, glucose

As before

Other additions:

   Fatty acids Linoleic acid, lipoic acid

  Nitrogen compounds

Hypoxanthine, thymidine, putrescine

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 growth factors

Insulin, transferrin, hydrocortisone, 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 ligandsmay 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 Media1. 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 requirementsvary 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.