—._.———-——-—
`
`-.'i|
`
`I-."‘-iI"I'-I’:
`
`r'
`
`s-l!‘.1.Ir-'!I r:_'h'r.1'.'ii-:'.i-;
`
`,_..-|._--,.
`
`-
`
`I; U ml P Practice
`
`European Journal of Hospital Pharmacy Practice
`
`Paediatric
`aerosol therapy
`
`K
`
`Age-related Maoular
`Degeneration
`
`Advances in treatment
`
`Practical aspects of biological therapy
`Cost-effectiveness of ranibizumab
`
`United Kingdom
`
`Transplantation
`. Steroid sparing strategies
`- Calcineurin inhibitor minimisation
`
`her
`anq. Belgium
`
`41:43am
`gamma Mun-2. rI-uzl-Jeltwlands
`Editor—inihlef
`..-. \a'iqnemn, FI-flnce
`
`rslnrle 1+
`
`1‘9 Del isiai. Mar; Fhaun, Mama
`
`
`
`Pharmacoeconomics
`a .2 movam or THE
`
`
`fingmifi NATIONAL
`
`%§3 LIBRARY OF
`_rvmm_ MEDICINE
`o Lenalidomide: a new therapy for multiple myeloma
`
`
`
`
`Dies
`
`HM Haer.
`
`Wound Management
`- Topical antimicrobial agents in burn care
`
`Officrai journai of the European association of Hospitai Pharmacists iEAHP}
`
`Page 1 of 11
`
`CSL EXHIBIT 1103
`
`Page 1 of 11
`
`CSL EXHIBIT 1103
`
`

`

`EJH P Practiee ill
`
`European Journal of HosPital Pharmacy Practice
`
`5in-P-€3o Official journal of the European Association of Hospital Pharmacists (EAHP)
`
` Contents
`a a i-
`
`49 'l‘ransplantalion ~ calcineurin
`52 Cost-effectiveness of genetic targeting ol'eancei' therapies
`58 Lenalidomide : m
`inhibitor minisation
`therapy For MM
`
`
`Feature
`
`Transplantation
`47 Steroid—sparing strategies in renal trinisplantaiion
`
`Pharmacoeconomics
`52 Establishing etist-el'lcctiveness ol' genetic targeting of
`
`49 Calcineurin inhibitor minimisation in renal transplantation
`
`cancer therapies
`54 What makes NICE tick?
`
`Haemto-oncology
`
`58 Lenalidomitle: a new therapy for multiple myeloma
`
`Wound Management
`
`62 Topical antimicrobial agents in hum care: a review
`
`
`Country Focus - UK
`
`41 Guild of Healthcare Pharmacists
`
`42 Clinical governance
`
`43 Exciting limes for education
`44 Medicine Intermalion services —
`
`knowledge for better care
`
`45 Medicine procurement practice
`
`46 Pharmaceutical Quality Assurance
`services
`
`Guidance for Authors of EjHP can be downloaded from www.ejhp.eu
`
`l
`
`Page 2 of 11
`
`Page 2 of 11
`
`

`

`E JHPPraas:
`
`Volume [4 ' ZOUBHSSUE it
`
`European Journal of H05pital Pharmacy Practice
`Official journal of the European Association of Hospital Pharmacists (EAHP)
`
`EJHP P
`
`€30
`
`Contents
`
`Medication Safety Forum
`
` .7}I
`
`.
`
`'
`
`b
`
`i.
`
`-
`
`t
`
`65 Aerosol them.
`P)“
`
`in hos iital:
`l
`
`I
`
`inediinric
`
`68 Inde Jendent drug information and I‘IuI‘rnncovi"ilnnce centres in ital
`l
`e
`:-
`
`:
`
`principles
`
`ClIMs
`
`WE “1-HT T0 HE A R FROM YO Ll
`
`: the Hamper!!! Jmuvm/ qf/Impffa/ Pkgrmm‘y — your journall
`
`Do you ever wonder how pharmacists Ji'rm'i {ieriuzm}. (ireeee. Siovuliu LllILl Spain can write such good
`[English in the l-JHI“? Would you like to tell us uhoui your won-L or opinions, hul I'eei your \\'I‘lliL‘I1 linelisli
`would let you down'.’
`
`The till-ll’ has :1 team oi native l-znglish—speziking L‘tlilftl's u'illi experience in lI|I\I}i1L1l pl'mnnue). medicine or
`science. They ure i'eudy Io help you express your ideas in professional
`lungungc uppropriute to _\our topic.
`They rain also suggest themes i'IJllLlIfl'N uould like in hear about and edit your uniele lo the l'ilell leneili.
`
`lil'L‘ esj‘icl‘icuce _\'r:Ll ham: us it hospital pliui'iuueist'. Sr: please tell lillll’
`Wlitil liiey eunuol supply is the real
`uhoul the dil'lercnee you ili'L" making to patient care. Simply ernuii us:11editoriulfirejliporerig-science“!ejlipoi'j.‘
`
`ulld your manuscript will he passed onto the right person.
` Inn-rested in
`
`b'i'opimmimwrit-nix for European Hospital Mini-moron is :i comprehensive compilation of
`uixJated :inicies previouslyr published in IL'JHP Practice. challenging sonic of the more. complex
`aspects of hiophummceutieul drugs. from chemistry to nomencialulc viii immunogenicily to risk
`management. Considering the increasing use of bitileehnologicully—inamifaetural drugs and [he
`recenl introduction of [he first follow—on veisions (biosimilarsi, the book is intended for hospital
`phunnucy practitioners as an aid in the process of evaluating these pi‘txincls.
`
`Editors: Schellckens ll. Vultu M;
`I3iophurlntlcetlticals for European I-Iospiiul Pharmacists - ISBN 973‘}th llfil‘illll
`
`l-‘or Iiill contenl inlonnul ion or order conditions. please conlalcl inarkeliligt'fl-'e_i|1p.org
`Tel: +32 41498953 - Fax: +32 E4531“ MK
`
`a
`-
`.
`.
`r
`,-
`m 'ls'" viii; li-s's‘i’g‘ps'ally-
`at the NLM and may be
`Subjenustom'rightLaws
`
`‘t
`.‘
`‘.
`_
`.
`t, ,
`s] l.t..l.vl\]:9lfll LR
`SAV [3 Mi /t
`
`h
`
`Page 3 of 11
`
`Page 3 of 11
`
`

`

`Eil H PPrac
`
`www.cjhp.eu
`
`t'ce
`
`ill
`
`’ Euro
`
`pean Journal of Hospital Pharmacy Practice
`
`l. Official journal of the European Association of Hospital Pharmacists (EAHP)
`
`0
`EJHP-P
`Volume l4 ' 2008ilssue 6
`
`
`
`EilHP is published bimonthly and mailed to more than M000 European hospital pharmacists, subscribers and key opinion leadeis in
`
`23 European countries and distributed at nnior international and national conferences. EjHP is aiaihble onii'ne {www.ejnpeu).
`
`Guest Authors:
`Anne Hiselius. Patrick Engelhardt. Dr jean-Daniel Hecq.
`Professor Paolo Lantctta. l‘rolessor Fiank G Holt. K
`Moerernans. Di- Vince S Thomas. L Annemans. Richard
`Cattell, Catherine Mooney. Susan Sanders. David Erskine.
`All-m Kai'r. Ian M Beaumont. Prolessor losep M Grinyo.
`Dr Helene Andersson, Prolessoi- Henrik Ekberg. DrValesca
`P Retel. Professor EJTh Rutgers, Dr MA joore. Professor
`WH van Harten. Dr Isaac A0 Odeyemi. Professor Pieter
`SDnnt-veid. Professor Antonio Pelumbo. Dr Gregory
`piondelot. Dr Jean Daniel Kaiser. Dr Veronique Noirez.
`Dr Hettie M Janssehs. Dr Elisabeth} Ruijgrok. Dr Harm
`AWM Tiddens. Dr Iienia Senesi. Dr Antonio Orsmi.
`0" R Di Tommaso. Dr F Sanita. F Harglotta. 5 Helena
`
`Author; for PRI Section:
`Professor Huuh Schellekens. Dr Nicole GM Huni‘eld.
`Dr Pauia FYpma. Dr Eisbem MWesterman. Dr Fiani: W
`Leebeek. Dr Danni] Touw
`
`Correspondents:
`Austria: Thomas Langebner
`Bfllxlulil: Dr Jean—Daniel Hem
`Croatia: Dr Vesna Vrta-Baéifi
`Czech Republic: Dr Marelc Liiéal‘
`Denmark: Linda Jeliery
`Estonia: Tiia Vals
`Finland: Dr Tiina Koskinen
`France: Dr Christian Cornette
`Germany: Dr Hans-Peter Lipp
`Greece: Desplna ldakridaki. Dr Ekaterini Tsiata
`Hungary: Monika Kis Stolgyemi
`Ireland; loan Peppard
`llaly: Dr Santolo Cotzoiino
`Latvia: Kristine \i'rubievska
`Lith uaril'a.‘ Blrute Va mnaviciene. Professor Vincen cis Veikutis
`Luxembourg: Patrick Engelhardt
`Netherlands: Dr Hans Dverdiel-t
`Norway.- Mareii Noi'dsveen Davies
`Poland: Anna Zaiaczkowska-Dutkiewifl
`Portugal:
`jot-go Bi-ochado
`s(H'lsizi: Svetlana Ristic
`Slovakia: Dr juraj Sykol'a
`SIt‘vvenia: Dr Tajdii Miharija-Gaia
`spalnzAna Maria Alvarez Diaz
`Sweden: Dr Srinivas Uppugundul‘l
`Switzerland: Dr Pascal Bonnabi'y
`Turkey: Mustaia Gonen. Dr Aydin Alper Saliin
`United Kingdomzli'ilma Gills
`
`Editor-In-Chlef:
`Professor Arnold G Vuito — av@ejhp.org
`
`Deputy Editor-inlehief:
`Dr Jean Vignerion - [v@e]hp.org
`
`Honorary Science Adviser:
`V'laln Fenton-May
`
`P u iJl isher:
`Lasia Tang - LT@ofhp,org
`
`Editors:
`Rob Cannon - rc@ejhp.org
`Michelle Gallather. M5: - mg@eihporg
`judith Martin. MRF’harmS - ili@e]hp.org
`Maris Nolan. RPII - in.nolan@eihp.org
`
`Science Editor:
`Diane Langlobfln.MRPf1arrnS. Cert Pharrn Prac - DL@ejhp.org
`
`Proiect Editor:
`Craig Stark.MD
`
`Editorial Assistant:
`Susanna Staplehurst - admln@ejhp.oi'g
`
`Marketing AssistantISUbscription:
`Evija Veirnere - marketrng®eihporg
`
`Production Assistant:
`Ann Dav-d . support@ejhp.org
`
`Science Assistant:
`Gaynor Ward - science@eihp.org
`
`Art Direction:
`Patriclr. Anthonls - art@eihp.org
`
`Subscription Rates 2009:
`Europe
`Individual:
`€23l
`HospitaIlUnive rsity:
`E495
`Como-ate:
`(562?
`Student:
`90
`
`Overseas
`€255
`if 5 I 9
`E GSi
`E | id
`
`1ndividua| and student subscriptions are subject to EMS
`VAT Belgian Government tax.
`
`EJHP Is published bi-nionthly. Subscription orders must he
`prepaid. Subscription paid is non-relundable. Cantact mar-
`keting@eiiip.org loi further inior'mntion.
`
`Change of address notices: both subscriber‘s old and
`new addresses should be sent to marketing@e]hp.org at
`least one month In advance.
`
`
`
`
`
`copyright@ppnie.eu I
`|P_ PM 0 Media
`
`O Pharma
`0 Publishing
`
`Published In Belgium by
`Pharma Publishing 8 Media
`Europe
`
`R
`
`0 P
`
`E
`
`-
`
`E
`
`U
`
`Printed by PPS s.a.
`
`Print Run: itooo— ISSN EJHP: ivainsas
`
`EJHP circulation is audited by EPA
`
`Worldwide — a not-ior—proiit organisation
`UBPAwen-wins
`who set the standard ior dioraughness.
`accuracy. transparency and timeliness in media and event audits.
`EPA serves 3.000 media properties.
`
`
`
`COPYRIGHT
`EJ 2008 Pharnia Publishing and Media Europe. All rights reserved.
`Materials printed in Ell-IF Practice and EJHP Science are covered
`by copyright. throughout the world and In all languages'ihe
`reproduction. transmission or storage in any form or by any
`Inc-ails either mechanical or electronic. Including subscription.
`photocopying. recording or through an information storage
`and retrieval system of the whole. 0r par-ts oi the. article in
`these publications is prohibited without consent of
`the
`publisher. The publisher retains the right to republish all con-
`trlhutions and Submitted materlals via the Internet and other
`media.
`
`Individuals may make single photocopies For personal non-
`commercial use without obtaining prior permission. Non-profit
`institutions such as hospitals are generally permitted to make
`copies of articles l'or rest-arch or teaching activities [including
`multiple copies For classrooms use} at no charge. prov-dad the
`institution obtains the prior consent From Pharma Publishing and
`Media Europe. For more information contact the Publisher.
`
`DISCLAI H ER
`While the publisher. editoris} and editorial board have taken
`every care with regard to accuracy ol editorial and advertise-
`ment contributionsthey cannot beheld responsible or in any
`way liable For any errors.omissinns or inaccuracies contained
`therem or for any consequeilcu arising there from
`
`Statements and opinions expressed in the articles and com-
`rnuniisitions herein are those of the authorfs) and do not
`necessarily reflect the views of the publisher, editol'is). 9mm.
`rial board and the European Association of Hospital
`Pharmacists iEAHPJ.
`
`Neither the publisher nor the editoris) nor the editorial
`board nor the EAHP guarantees. warrants. or endorses any
`product or service advertised In this publication. nor do they
`guarantee any claim made by the manufacturer ol such
`Claims: When claims for undelivered or damaged issues
`product or service. Because of rapid advances in the medical
`are made within lour months of publication ol the iSiiue.a
`sciences. in particular. Independent verification oF diagnoses
`complimentary replacement issue will be provided.
`and drug dosages should be made.
`———————
`
`EAHP onice:
`3 Rue Abbe Cuypers. Bv |D40 Brussels. Belgium
`El 1 *31 2 IN l6322i19 - Far. : +32 2 il‘l'fllo
`““CQeahpeii - wwwealipeu
`
`EJHP Editorial Office:
`Postbus moo I. emu MOL. Belgium
`Tel :+31 04939er — Fax : +32 i4 583049
`LE ditorial@e|h p.o rg - www.cihp.eu
`
`Page 4 of 11
`
`Page 4 of 11
`
`€
`€
`

`

`PracticeResearch & Innovation
`
`Why some proteins have sugars? Epoetins, from
`alfa to zeta
`
`Professor Huub Sohellekens. MD. PhD
`
`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 procesa of glycosyiation and its biological significance is reviewed. The differences in glycosylation of the various
`erythropoietins and their possible clinical significance are also discussed.
`
`stwonos
`
`Bioslmilars. epoetlns, giycosylation. therapeutic proteins
`
`INTRODUCTION
`
`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. egCOSylation 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.
`
`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 glycosyiation
`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
`
`erytl‘vopoietin 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 giycosylated [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 glycosylatlon.
`
`GLYCOSYLA‘I‘ION OF 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 Schelielcens. MD. PhD
`Departments of Pharmaceutical Sciences and Innovation Studies
`Utrecht University
`PO Box 80082
`3508 TB Utrecht. The Netherlands
`Tel: + 31 30 2536973/253?305
`Fax: + 31 30 251?839
`h.schel|ekens@uu.nl
`
`EJ H P Practice - Volume14 " 200316
`
`
`
`EIHPI 1|.“ r .Illvimi |--.--|rn |lri| flu-i '4IIIf-I-.1|1 AT... ..|I|- .u- II t ind-ital I'Jmnu-i- 1-.|
`
`rl N 1",!
`
`
`
`www.cjhpeu 2 9
`
`Page 5 of 11
`
`Page 5 of 11
`
`

`

`PracticeResearch & Innovation
`
`Table 1: Functions Of the glyco component Of
`glycoproteins
`
`Protein folding
`
`Protein trafficking and targeting
`
`Protein targeting
`Ligand recognition
`
`Ligand binding
`
`
`
`Biological activity
`
`Stability
`Pharmacokinetics
`
`lmmunogenicity
`
`categories depending on the way the glycan is linked:
`O—linked. N—Iinked. C-linked and amide linked.
`
`o-LINKED vacosmnon
`
`O-linked giycosylation 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-tum, 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 GLYCOB‘I'LM’lON
`
`This type of glycosylaticn is sequence dependent and only
`occurs at a specific combination of three amino acids [6].
`MostOftheseso-caliedAsn-X—Serfl'hrconsensussequences
`are, however. non~glycosylated. The secondary structures
`are important to determine whether giycosylation occurs.
`Glycosylation ls performed before the proteins
`fold
`and acquire their
`final
`three dimensional structures.
`N»linked glycosylation starts with the attachment of an
`oligosaccharide consisting of two N-acetylglucosamine
`lGlcNAc}. 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
`glycosyiation. Eventually GlcNAc sugars may be attached
`resulting in complex type N—glycosylation. This type of
`glycosylation is the main cause of the heterogeneity Of
`glyCOprotelns and the generation of several glycoforms.
`
`C-LINKED vacovaAnoN
`
`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.
`
`Table 2: Analytical tools to study glycosylation HF-‘LC of released Oligosaccharides
`
`Capillary electrophoresis
`Mass spectrometry
`
`Amos-LINKED GPl-ANCHORED PROTEINS
`
`These are proteins anchOred to membranes by covalent
`linkage to glyc0sylphosphatidyiinositol (GPIJ [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.
`
`Funcnoss or me GLYCOCOMPONENT OF GLYCOPHOTEINS
`PROTEIN FOLDING
`
`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 EFl 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 oi 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 3D structure. this occurs in
`cases where the glycan moiety only plays a role in the
`folding process without being necessary for maintaining
`the structure.
`
`Pnonzm rsnrncxms AND TAHGETING
`
`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
`01 the most
`important reOOQnition 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 cellnto-matrix
`interactions and proteln-cell
`reOOQnilion. As lectins are
`differentially expressed. tissues have varying affinities for
`
`30 EJHPPraclice - Volume14 - zoos/s
`
`www.e]hp.eu
`
`Page 6 of 11
`
`Page 6 of 11
`
`

`

`PracticeResearch & Innovation
`
`specific glycoproteins. For example. glucocerebrosidase
`(G
`l. which is used to treat Gaucher's disease. has a
`specific glycan structure. targeting it to manncse-binding
`lectins on macrophages of the liver. where the enzyme
`catalyses the hydrolysis of the glycaltpid glucocerebroside
`to ceramide and glucose [12}.
`
`LIGAND smomc
`
`Giycosylatlon modulates the interaction of glycoproteins
`with specific receptors. For example. the Fc part of the
`heavy chains of immunoglobulins contain a single N-linked
`glycosylation site essential for interactions with Fe recep—
`tors {FcFli. which are present on cells such as natural killer
`cells and macrophages [13], This interaction with Fth
`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 immunoglobuiins exhibit
`severely impaired ADCC and CDCr
`
`BIOLOGICAL acnvrrv
`
`Glycosylation is also important for biological activity as
`exemplified by the glycoprotein hormones TSi—l {thyroid
`stimulating hormone}. FSH (follicle stimulating hormone}.
`LH (Iuteinising hormone) and hCG [human chorionic
`gonadctrophini. N—linked glycosylation has only a minor
`effect on receptor binding but is critical for bioactivity. In
`fact. if these hormones lack N—Iinked glycans. they act as
`antagonists [l4]. Glycans may also be important for the
`oligomerisatiori necessary for biological activity. as has
`been shown for gastric mucin which protects the stomach
`from chemical and other damage.
`
`STABitmr
`
`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 oiigosaocharides may protect them
`from proteases or antibodies resulting in an enhanced
`half—life and bioavailability [15].
`
`PHARMACOIGNEI’ICS
`
`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 [1 6]. Hyperglycosylation may increase
`
`plasma halt-life. although carbohydrates can enhance
`lectin-medlated clearance. eg. glycoproteins with a terminal
`mannose are cleared through the reticulo—endothelial
`system by receptors with a high affinity for manncse. 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.
`
`‘
`Immunoesmciw
`Giycan 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. ODE. 0022 and
`CD28. The non-cellular parts of the immune system Such
`as C—reactive protein.
`the immune giobulins and many
`of the interieukins and cytokines are also dependent on
`glycosyiation for their proper function [1?].
`
`Carbohydrates can also be the cause of an immunogenio
`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. Mucopclysaccharides are also
`major
`immunogenic components of some vaccmes.
`Glycosylation may be necessary for the complex forma—
`tion needed for immunogenicity. as has been shown for
`the hepatitis B surface antigen. Glycan structures have
`also been implicated in the allergic reaction to monoclona
`antibodies [181. The autoimmune response to cogtaigen
`which is important forthe development of arthritis is
`r ven
`by glycosylation of the T—cell epitope involved [19].
`
`However. carbohydrates have never been shown to form
`'
`'
`'
`'
`'
`. Glycans.
`ito es of therapeutic glycoproteins .
`immunogenlc 3p
`p
`ffects. which initiate an immune
`however. may have indirect e
`_
`‘
`response to the therapeutic protein. Glycoprcteins produced
`in prokaiyotic host cells. and therefore lacking carbohygatisa
`havean increasedimmunogenicityeitherbecauseofar (ssh
`solubility or through exposed epitopes normally protect
`y
`glycans [20].
`
`ELATED TO ABNORMAL GLYCOSYLATION
`giggiaygosyiation is the most important way to’oonvey
`information in biological systems.
`it
`ls-no surprise that
`abnormal glycosylation is increasmgly being recognised as
`an important factor in the pathogeness of diseases. Deli-
`cient. abnormal. and hyper-gtycosylation, both acquired
`and genetic. have all been identified as causes of disease
`symptoms. Some excelient reviews have been published
`recently discussing glycosylation disorders.
`therefore
`
`EJHPPractice - Volume 14 - spears
`
`
`
`EJHP .-. IIi-.- rJlilf lull
`
`li .ui mil oi livu E'Inll'ilJI-ull A mnmii-u. ..Il i-. Mimi Ph]tl11._v'1-. ii ni-Il I)
`
`
`
`www.cihp.eu 3 1
`
`Page 7 of 11
`
`Page 7 of 11
`
`

`

`PracticeResearch & Innovation
`
`only a few examples are mentioned here to illustrate how
`glycosylation affects biological function 121. 22].
`
`The most dramatic diseases caused by abnormal gly-
`cosylation are the congenital disorders of glycosylation
`(CDGJ, 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.
`
`The first CDG described was Inclusion-cell disease (I—cell
`disease or mucolipidosis II). a storage disease of lyso-
`somes caused by inhibition of the mannose-B-phosphate
`modification of N~linked glycosylation ta signal for the traf-
`ticking of proteins to the lysosome). The most prevalent
`disease is CDG type is. 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 glycosyiaticn site.
`e.g.
`in the interferon receptor. IFNAFi2. which abolishes
`the cellular response to interferon site [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 IgG figs-GO). This lack of terminal galac—
`tose in the branches of the N-Iinked 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 reaponse. The exposed
`N~acetylglucosarnine residues. which are no longer cov-
`ered by galactose. also interact with MBL (mannose bind-
`ing Iectln) inducing complement activation.
`
`ABNORMAL vacovaanou 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
`lesions by isoeiectric focusing is used as a general tool
`for the diagnosis of CDG [25].
`
`Pnooucme GLYCOSYLATED THERAPEUTIC PROTEINS
`
`Although some glycoproteins. such as G-CSF {granuiocyte
`colony~stimulating factor] and human interferon alfa 2. do
`not need glycan for their therapeutic activity and are pro—
`duced successfully in E. coil. 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]. N30 cells produce more
`heterogeneous proteins. In addition. NSD 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 Pichia bacteria
`may produce up to 30 9.4.. 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 glycosyiation
`and thereby the therapeutic Drofile 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 erythrcpoietin loses biological
`activity when heated. while the giycosylated form remains
`active [28]. Also glycosylated interferon beta is more
`heat-stable than the deglycosylated form [29].
`
`Natural glycosylation can be modified to enhance the
`therapeutic efficacy of biopharmaceuticals. The best exam-
`ple is that of the hyperglycosylated form of erythropoietin.
`darbepoletin site. 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-Iinked glycosylation
`sequence extension which increases the serum half-life
`fourfold because of reduced renal clearance of the protein.
`Modification ofglycosylation can also be used to enhancethe
`effector functions of monoclonal antibodies. e.g. increased
`ADCC activity was achieved by using various glycosylation
`pathway inhibitors during production [30]. Glycosylation
`can also influence drug targeting. Glycoproteins containing
`terminal galactose or N-acetylgalactosamine are specifi-
`cally cleared by receptors in the liver [31].
`
`32 EJHPPractice - yam-314 - 200%
`
`www.cjhpeu
`
`Page 8 of 11
`
`Page 8 of 11
`
`

`

`PracticeResearch & Innovation
`
`Emerge op GLYCOSYLATED THERAPEUTIC PROTEiNS
`MONOGLONAL mneonres
`
`All approved therapeutic monoclonal antibodies are
`derived from immunoglobulin G. mainly lgGi.
`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. Giycosylation is essential for the structure and
`function of the F0. 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 lCAMPATi—i—t H). 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.
`
`Not only are the Fc regions of monoclonal antibodies
`glycosyiated 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-
`pie. an anti—aliat. 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].
`
`Enwriaoeorenrts
`
`Erythropoietins are currently the most widely used thera-
`peutic glycoproteins. The core protein contains 165 amino
`acids but 40% of the final molecular weight
`is sugar.
`consisting of three linked N-giycosyiation sites at Asn24.
`38 and 83 and one O~iinked site at serine 126. The
`
`heterogeneity of erythropoietins is caused by variations in
`core structures and sialic acid between the different sites
`
`and also between different isotonns. Glycosylation is impor-
`tant not only for the structural integrity and the secretion of
`the protein by the producing cells. but also for its biological
`activity. Removal of at
`least
`two N-linked glycosylation
`sites by site-directed-mutagenesis leads to inhibition of
`the production of erythropoietin by cells [34]. Removal of
`iii—linked glycans increases the in vitro activity, but abolishes
`the in vivo activity. Adding N-giycosyiation sites decreases
`in vitro receptor b