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`PracticeResearch & Innovation
`
`Why some proteins have sugars? Epoetins, from
`alfa to zeta
`
`Professor Huub Schellekens, MD, 3hD
`
`ABSTRACT
`
`Recently the first biosimilar erythropoietins were introduced. The main differences between biosimilars and the original
`products are related to glycosylation. Glycans are the most information dense structures in nature. However, the relation—
`ship between glycosylation and function is not yet completely understood. in this article our current knowledge about
`the process of glycosylation and its biological significance is reviewed. The differences in glycosylation of the various
`erythropoietins and their possible clinical significance are also discussed.
`
`www.ejhp.eu 2 9
`
`Glycoproteins are the most diverse natural polymers [1—4].
`About 2% of human genes are involved in glycosylation.
`With four different types of glycosylation involving nine
`different sugars with varying links to either the protein or
`another sugar, and the size of the glycosylation varying
`from a single sugar to a complex branched glycan struc-
`ture, the possibilities for variations in glycan structure are
`endless. Indeed, glycosylation is the most complex biolog—
`ical signalling system. The amount of information stored in
`
`glycoproteins far exceeds the storage capacity of DNA.
`Glycosylation is also the main reason for the heterogeneity
`of proteins, since it may differ from site to site within the
`protein molecule, from molecule to molecule, from cell to
`cell and finally from tissue type to tissue type.
`
`KEYWORDS
`
`Biosimilars, epoetins, glycosylation, therapeutic proteins
`
`INTRODUCTION
`
`Understanding of the complexity of glycosylation and its
`biological functions is still far from complete (Tables 1 and 2).
`especially since there is no template for glycosylation
`as there is for RNA and protein synthesis. On the other
`hand, this makes glycosylation a faster and more flexible
`way to adapt to external pressures and changes in the
`environment. But why, for instance, different isoforms of
`
`erythropoietin exist, which differ in their biological function,
`is
`still under fierce debate. However,
`the increasing
`recognition of diseases related to abnormal glycosylation
`is both proof of its biological significance and an aid to
`the understanding of its biological functions.
`In addition,
`proteins with abnormal glycosylation are increasingly being
`used as disease markers.
`
`Proteins, such as growth factors, cytokines, hormones,
`monoclonal antibodies and others are becoming an
`important part of our therapeutic arsenal. About 50% of
`these therapeutic proteins are glycosylated [1]. Recently,
`a number of biosimilars (or follow-on biologics in the US)
`have been introduced which differ in their glycan struc—
`ture. So it is important for prescribers to understand the
`biological importance of glycosylation and therefore this
`paper will review the current understanding of the function
`of glycosylation.
`
`GLYCOSYLATION 0F PROTEINS
`
`Proteins are glycosylated in the endoplasmic reticulum
`(ER) and Golgi apparatus by glycosidases and glyco-
`syltransferases. Glycoproteins can be divided into four
`
`CONTACT FOR CORRESPONDENCE:
`
`Professor Huub Schellekens, MD, PhD
`Departments of Pharmaceutical Sciences and Innovation Studies
`Utrecht University
`PO Box 80082
`3508 TB Utrecht, The Netherlands
`Tel: + 31 30 2586973/2537305
`Fax: + 31 30 2517889
`h.schellekens@uu.nl
`
`E] H P Practice - Volume 14 ' 2008/6
`
`EJHP i
`
`. Iii-rerir ml Jfllillitll 01 iii? i‘tiropvzziii A'fi’JClrIt/ill oi l’luupilul l‘iitiiniti'nfil'; (FAHP)
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`Page 5 of 11
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`

`

`PracticeResearch & Innovation
`
`Table 1: Functions of the glyco component of
`glycoproteins
`
`Protein folding
`
`Protein trafficking and targeting
`
`Protein targeting
`Ligand recognition
`Ligand binding
`
`www.cjhp.eu
`
`Glycosylation and protein folding are closely related
`processes in the endoplasmic reticulum which is the first
`station on the track of protein excretion by cells [10].
`The ER contains the enzymes necessary for oligosac—
`charide attachment to proteins and glucose trimming
`and the chaperone proteins necessary for folding. The
`quality control for the exact glycosylation and folding
`also occurs in the ER and the misfolded products are
`removed to prevent clogging. Glycosylation starts as
`soon as the nascent polypeptide chains reach the ER.
`The attached glycans confer solubility to the polypeptide
`and also recruit the chaperone proteins which are nec—
`essary for correct folding. Although in numerous cases
`glycosylation is essential for protein structure, preven—
`tion or modification of glycosylation of many proteins
`has no effect on synthesis or folding. Likewise, there are
`examples of the removal of oligosaccharides from a pro-
`tein without an effect on the 8D structure, this occurs in
`cases where the glycan moiety only plays a role in the
`folding process without being necessary for maintaining
`the structure.
`
`Table 2: Analytical tools to study glycosylation
`HPLC of released oligosaccharides
`Capillary electrophoresis
`[ Mass spectrometry
`
`AMIDE-LINKED GPl-ANCHORED PROTEINS
`
`These are proteins anchored to membranes by covalent
`linkage to glycosylphosphatidylinositol (GPl) [8, 9]. These
`proteins are exclusively located on the extracellular side of
`the plasma membrane and form a diverse family of mole—
`cules including membrane—associated enzymes, adhesion
`molecules and other glycoproteins. An example of a GPI—
`anchor is CD59 which protects cells from complement—
`mediated damage.
`
`FUNCTIONS OF THE GLYCOCOMPONENT OF GLYCOPROTEINS
`PROTEIN FOLDING
`
`PROTEIN TRAFFICKING AND TARGETING
`
`Glycosylation may also be important for directing proteins
`to the correct cellular compartment after excretion by the
`ER. Proteins intended for lysosomal function often carry a
`mannose-B-phosphate as a signalling residue [11]. One
`of the most
`important recognition systems throughout
`nature is the interaction between lectins and glycoproteins.
`Lectins are carbohydrate binding proteins which play a role
`in host—pathogen interaction, cell-to-cell and cell—to-matrix
`interactions and protein-cell recognition. As lectins are
`differentially expressed, tissues have varying affinities for
`
`Biological activity
`
`Stability
`Pharmacokinetics
`
`[i_mmunogenicity
`
`J
`
`categories depending on the way the glycan is linked:
`O-Iinked, Nelinked, C-Iinked and amide linked.
`
`O-LINKED GLYCOSYLATION
`
`O—linked glycosylation occurs to specific serine or threonine
`residues, but a consensus sequence has not been identi—
`fied [5]. Apparently secondary structural elements, such as
`a beta~turn, define whether these amino acid residues will
`
`be glycosylated. This type of glycosylation can result in the
`production of mucin—type glycoproteins which, for example,
`are involved in inflammation but can also be found in thera-
`
`peutic proteins such as erythropoietin and interferon alfa.
`
`N-LINKED GLYCOSYLATION
`
`This type of glycosylation is sequence dependent and only
`occurs at a specific combination of three amino acids [6].
`Most of these so-called Asn—X- Serffhrconsensus sequences
`are, however, non-glycosylated. The secondary structures
`are important to determine whether glycosylation occurs.
`Glycosylation is performed before the proteins
`fold
`and acquire their
`final
`three dimensional
`structures.
`N-Iinked glycosylation starts with the attachment of an
`oligosaccharide consisting of two N—acetylglucosamine
`(GlcNAc), nine mannose and three glucose molecules, The
`glycan structure is then trimmed by the removal of glucose
`and mannose residues leading to a high-mannose type of
`glycosylation. Eventually GlcNAc sugars may be attached
`resulting in complex type N-glycosylation. This type of
`glycosylation is the main cause of the heterogeneity of
`glycoproteins and the generation of several glycoforms.
`
`C-LlNKED GLYCOSYLATION
`
`In this type of glycosylation, a sugar-like mannose is
`linked to the protein through a carbon—carbon bond [7].
`This type of glycosylation mainly occurs in proteins con-
`taining sequences necessary to bind to other proteins
`such as complement factors, receptors for cytokines and
`hormones.
`
`30
`
`Practice ' Volume 14 ' 2008/6
`
`Page 6 of 11
`
`

`

`PracticeResearch & Innovation
`
`plasma half—life, although carbohydrates can enhance
`lectin-mediated clearance, e.g. glycoproteins with a terminal
`mannose are cleared through the reticuloendothelial
`system by receptors with a high affinity for mannose. The
`O-linked glycosylation of glycoproteins like FSH and others
`has no effect on receptor binding or biological activity but
`has a dramatic effect on in vivo half—life and bioactivity.
`
`_
`IMMUNOGENICITY
`Glycan structures play an important role in cell—to-cell
`communication and protein cell
`interaction, which are
`essential for the immune system to function. Activation of
`immune cells is also dependent on glycosylation through
`receptors such as the T-cell receptor, CD8, CD22 and
`CD28. The non-cellular parts of the immune system such
`as C-reactive protein, the immune globulins and many
`Of the interleukins and cytokines are also dependent on
`glycosylation for their proper function [17].
`
`specific glyCOproteins. For example, glucocerebrosidase
`(GBA), which is used to treat Gaucher’s disease, has a
`specific glycan structure, targeting it to mannose-binding
`lectlns on macrophages of the liver, where the enzyme
`catalyses the hydrolysis of the glycolipid glucocerebroslde
`to ceramide and glucose [12].
`
`LIGAND BINDING
`
`Glycosylation modulates the interaction of glycoproteins
`with specific receptors. For example, the Fc part of the
`heavy chains of immunoglobulins contain a single N~|inked
`glycosylation site essential for interactions with Fe recep—
`tors (FCR), which are present on cells such as natural killer
`cells and macrophages [18]. This interaction with FOP]
`mediates effector functions, including antibody—dependent
`cell cytotoxicity (ADCC) and complement—dependent cyto-
`toxicity (CDC). The glycosylation of the Fc part is essen—
`tial because non—glycosylated immunoglobulins exhibit
`severely impaired ADCC and CDC.
`
`BIOLOGICAL ACTIVITY
`
`Glycosylatlon is also important for biological activity as
`exemplified by the glycoprotein hormones TSH (thyroid
`stimulating hormone), FSH (follicle stimulating hormone),
`LH (Iuteinising hormone) and hCG (human chorionic
`gonadotrophin). N-linked glycosylation has only a minor
`effect on receptor binding but is critical for bioactivity.
`in
`fact, if these hormones lack N—linked glycans, they act as
`antagonists [14]. Glycans may also be important for the
`oligomerisation necessary for biological activity, as has
`been shown for gastric mucin which protects the stomach
`from chemical and other damage.
`
`STABILITY
`
`www.ejhp.eu 3 1
`
`Carbohydrates can also be the cause of an lmmunogenic
`response. Carbohydrates determine the antigenic struc—
`tures of the ABO (H) and related Lewis blood groups and
`humans have natural antibodies to these glycan structures
`and others which occur in nearly all non—primate speCIes,
`including microorganisms. Mucopolysaccharides arealso
`major
`immunogenic components of some vaccines.
`Glycosylation may be necessary for the complex forma—
`tion needed for lmmunogenicity, as has been shown for
`the hepatitis B surface antigen. Glycan structures haveI
`also been implicated in the allergic reaction to monoclona
`antibodies [18]. The autoimmune response
`collagen
`which is important for the development of arthritis is driven
`by glycosylation of the T—celi epitope involved [1 9].
`
`Glycosylation influences the stability of proteins in different
`ways and, as has been discussed, may be important for
`the structural integrity of glycoproteins. But glycosylation
`also enhances solubility and thermal stability as well as
`preventing aggregation and loss of activity. Moreover, the
`‘coating’ of proteins by oligosaccharides may protect them
`from proteases or antibodies resulting in an enhanced
`half-life and bioavailability [15].
`
`However, carbohydrates have never been shown to form
`lmmunogenic epitopes of therapeutic glycoproteins. Qlycans,
`however, may have indirect effects, which initiate an iménun:
`response to the therapeutic protein. Glycoproteins prod uitce
`in prokaryotic host cells, and therefore lacking carbohy ra esCi
`have an increased immunogenicity either because ofa reduce
`solubility or through exposed epitopes normally protected by
`glycans [20].
`
`PHARMACOKINETICS
`
`For proteins such as hormones and growth factors,
`which are active at a distance from the site of production,
`pharmacokinetic properties are important for their biological
`function. These are affected by many factors, such as
`molecular size and charge, which may depend on the glycan
`structures. Sialic acid,
`in particular, contributes to the net
`negative charge and improves the half-life of glycoproteins
`such as erythropoietin [16]. Hyperglycosylation may increase
`
`DISEASES RELATED To ABNORMAL GLYCOSYLATION
`Since glycosylation is the most important way to convey
`information in biological systems,
`it isno surprise that
`abnormal glycosylation is increasingly being recognised as
`an important factor in the pathogeneSis of diseases. Defi-
`cient, abnormal, and hyper—glycosylation, both acquired
`and genetic, have all been identified as causes of disease
`symptoms. Some excellent reviews have been published
`recently discussing glycosylation disorders,
`therefore
`
`EJ HP Practice - Volume 14 ' 2008/6
`
`EJHP i: the Oiiiual Juumal oi the Eulopuvifl Aunucialion oi Ho'mlidi Pituirnuniul‘; (EAHF‘)
`
`Page 7 of 11
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`

`

`PracticeResearch & Innovation
`
`only a few examples are mentioned here to illustrate how
`glycosylation affects biological function [21, 22].
`
`The most dramatic diseases caused by abnormal gly—
`cosylation are the congenital disorders of glycosylation
`(CDG),
`for
`instance, glycosidase deficiencies such as
`Gaucher‘s, Neumann—Pick type C and Tay-Sachs dis—
`ease. Although glycosidases play a role in the maturation
`of glycoproteins by trimming the glycan structures, their
`main tasks are degrading glycoproteins in the lysosomes.
`Consequently the main manifestations of these diseases
`are cellular storage disorders associated with severe
`malformations and/or metabolic abnormalities. At
`the
`
`moment approximately 20 genes and about 100 allelic
`variants have been identified; however, it is still unknown
`how many CDG type diseases exist and how common
`they are.
`
`PRODUCING GLYCOSYLATED THERAPEUTIC PROTEINS
`
`Although some glycoproteins, such as G-CSF (granulocyte
`colony-stimulating factor) and human interferon alfa 2, do
`not need glycan for their therapeutic activity and are pro
`duced successfully in E co/I', most biopharmaceuticals
`need to be produced in eukaryotic cells which have the
`machinery to add glycans. Different host cells, such as
`the hybridoma cell—line NSO, a mouse hybrid myeloma cell
`line SP2/O or Chinese hamster ovary (CHO) cells are used
`to produce biopharmaceuticals and the cell type has a major
`influence on glycosylation [26]. N80 cells produce more
`heterogeneous proteins. In addition, NSO cells, being cancer
`cells, contain a higher amount of N—glycolylneuraminic acid,
`which is absent in CHO cells. CHO cells are used most fre—
`
`quently to produce glycoproteins. Up to 5 g/L can be pro—
`duced in CHO cells, while yeasts such as Pic/via pastor/s
`may produce up to 30 g/L, but usually with a high mannose
`content, which can result in a short in vivo half—life and pos—
`sible loss of efficacy. Glycoengineering is, however, used to
`introduce the human enzymes in yeast, which is necessary
`for the production of glycoproteins with a more human—like
`glycan structure [27]. Culture conditions such as the com—
`position of the culture media, temperature, cell density, and
`purification influences the heterogeneity of glycosylation
`and thereby the therapeutic profile of the final product.
`
`Glycosylation is important not only for the manufacture,
`but also for maintaining the stability of therapeutic proteins
`since glycosylation protects the therapeutic protein from
`proteolysis and increases its temperature stability. For
`example, deglycosylated erythropoietin loses biological
`activity when heated, while the glycosylated form remains
`active [28]. Also glycosylated interferon beta is more
`heat—stable than the deglycosylated form [29].
`
`www.ej hp.eu
`
`Natural glycosylation can be modified to enhance the
`therapeutic efficacy of biopharmaceuticals. The best exam—
`ple is that of the hyperglycosylated form of erythropoietin,
`darbepoietin alfa, which contains additional N—linked
`glycosylation sites. This
`increases the serum half—life
`threefold and enhances biological activity, resulting in less
`frequent parenteral dosing. Another example is a modified
`FSH which contains a consensus N-linked glycosylation
`sequence extension which increases the serum half—life
`fourfold because of reduced renal clearance of the protein.
`Modification of glycosylation can also be used to enhance the
`effector functions of monoclonal antibodies, e.g. increased
`ADCC activity was achieved by using various glycosylation
`pathway inhibitors during production [80]. Glycosylation
`can also influence drug targeting. Glycoproteins containing
`terminal galactose or N—acetylgalactosamine are specifi—
`cally cleared by receptors in the liver [81].
`
`The first CDG described was Inclusion—cell disease (l-cell
`disease or mucolipidosis ll), a storage disease of lyso-
`somes caused by inhibition of the mannose—B—phosphate
`modification of N—linked glycosylation (a signal for the traf-
`ficking of proteins to the lysosome). The most prevalent
`disease is CDG type ta, which is based on mutations in
`the PMM2 gene. This gene codes for an enzyme that is
`essential
`for the initiation of N-glycosylation. However,
`CDG are not only restricted to the enzymes responsible for
`the adding or removing of sugar moieties. Mutations have
`been described which lead to an extra glycosylation site,
`e.g.
`in the interferon receptor,
`IFNAR2, which abolishes
`the cellular response to interferon alfa [23].
`
`A striking example of an acquired disease caused by
`abnormal glycosylation is rheumatoid arthritis [24]. Disease
`activity is highly associated with the presence of non—
`galactosylated lgG (lgG—GO). This lack of terminal galac-
`tose in the branches of the N—linked glycans leads to a less
`rigid structure of the lgG molecule that is more prone to
`aggregation. The lack of galactose also exposes epitopes,
`which induce
`an anti-lgG response. The exposed
`N—acetylglucosamine residues, which are no longer cov—
`ered by galactose, also interact with MBL (mannose bind—
`ing lectin) inducing complement activation.
`
`ABNORMAL GLYCOSYLATION AS DISEASE MARKERS
`
`With the increasing identification of disease conditions
`related to abnormal glycosylation, there is also a need for
`disease markers.
`lgG—GO is recognised as a predictive,
`specific and sensitive marker for the development and
`activity of rheumatoid arthritis. The identification of ferritine
`isoforms by isoelectric focusing is used as a general tool
`for the diagnosis of CDG [25].
`
`32
`
`H P Practice - Volume’l4 ' 2008/6
`
`Page 8 of 11
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`

`

`PracticeResearch & Innovation
`
`the net result being an increased clinical efficacy. Highly
`branched and highly sialylated N—linked glycosylation is
`associated with a higher biological activity. Removal of the
`terminal sialic acids exposes the galactose residues, which
`are recognised by the hepatic galactose receptors resulting
`in increased clearance.
`
`As with other biopharmaceuticals, the glycosylation pattern
`is dependent on the host cell, production and purifica—
`tion methods. This was confirmed by the introduction of
`so-called biosimilar products in Europe, made possible by
`the expiration of the original erythropoietin patent and the
`submission of a request for marketing authorisation with
`more limited documentation compared with innovative
`therapeutic proteins. According to European regulations.
`biosimilars need to be extensively compared with the original
`products, including clinical trials [35-7]. Many factors influ—
`ence glycosylation including the host cell used, the math
`ods used to genetically modify the cell and the downstream
`processing all of which differ between different manufactur—
`ers, and the main differences between the biosimilars and
`the original products concern glycosylation.
`
`In Table 3, a description of the different biosimilar erythro-
`poietins can be found. Although it is not a biosimilar, epoe—
`tin delta is included, because the same issues concerning
`glycosylation apply. The data concerning biosimilars derive
`from the European Public Assessment Report
`(EPAR),
`which is published as soon as a drug is admitted to the
`European market [38-41].
`
`One of the major issues concerning the introduction of
`biosimilar erythropoietin is the possible occurrence of pure
`
`
`
`
`
`
`
`Table 3: Glycosylation and clinical efficacy of different
`epoetins
`INN RegulatoryBrand names GlyCOSY'afiO"
`Claimed to be
`more human
`
`All approved therapeutic monoclonal antibodies are
`derived from immunoglobulin G, mainly lth. Immu—
`noglobulin G consists of two light and two heavy chains,
`forming two identical specific antigen—binding sites and an
`Fc region which carries functions such as phagocytosis,
`antibody—dependent cellular cytotoxicity and complement
`activation. Glycosylation is essential for the structure and
`function of the Fc. The glycoform profile of monoclonal
`antibodies produced in CHO, N80 or SP2/O cells also
`shows heterogeneity, which can vary depending on the
`cell type, and specific clone used and, like other biophar-
`maceuticals,
`is dependent on the production and purifi-
`cation conditions. A good example is the production of
`humanised anti—CD52 (CAMPATH-fH), which is used for
`the treatment of lymphoma,
`leukaemia and rheumatoid
`arthritis. The biological activity, as measured by ADCC,
`depends on the cell source of the monoclonal antibody.
`To optimise the activity of monoclonal antibodies, engi—
`neered host cells are used which has resulted in glyco-
`forms with greatly improved ADCC.
`
`www.cjhp.eu 3 3
`
`EXAMPLES OF GLYCOSYLATED THERAPEUTIC PROTEINS
`MONOCLONAL ANTIBODIES
`
`Not only are the Fc regions of monoclonal antibodies
`glycosylated but also the antigen binding variable regions.
`The functional significance of this has not been fully evalu-
`ated, but data suggests that it has various influences on
`antigen binding, both positively and negatively. For exam—
`ple, an anti-alfat, 6-dextran antibody had a 15-fold higher
`affinity for antigen when a glycan was attached at Asn—58
`within the variable region [32]. Altering the location of
`glycans within the variable region by site-directed muta-
`genesis can completely inhibit antigen binding by steric
`blocking of the interaction by the sugar [33].
`
`ERYTHROPOIETINS
`
`Erythropoietins are currently the most widely used thera-
`peutic glycoproteins. The core protein contains 165 amino
`acids but 40% of the final molecu