REVIEWS
`
`Effects of Glycosylation on the Stability of
`Protein Pharmaceuticals
`
`RICARDO J. SOLA´ , KAI GRIEBENOW
`
`Laboratory for Applied Biochemistry and Biotechnology, Department of Chemistry, University of Puerto Rico,
`Rı´o Piedras Campus, Facundo Bueso Bldg., Lab-215, PO Box 23346, San Juan 00931-3346, Puerto Rico
`
`Received 21 December 2007; revised 14 May 2008; accepted 19 June 2008
`
`Published online 25 July 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21504
`
`In recent decades, protein-based therapeutics have substantially expanded
`ABSTRACT:
`the field of molecular pharmacology due to their outstanding potential for the treatment
`of disease. Unfortunately, protein pharmaceuticals display a series of intrinsic physical
`and chemical instability problems during their production, purification, storage, and
`delivery that can adversely impact their final therapeutic efficacies. This has prompted
`an intense search for generalized strategies to engineer the long-term stability of
`proteins during their pharmaceutical employment. Due to the well known effect that
`glycans have in increasing the overall stability of glycoproteins, rational manipulation of
`the glycosylation parameters through glycoengineering could become a promising
`approach to improve both the in vitro and in vivo stability of protein pharmaceuticals.
`The intent of this review is therefore to further the field of protein glycoengineering by
`increasing the general understanding of the mechanisms by which glycosylation
`improves the molecular stability of protein pharmaceuticals. This is achieved by pre-
`senting a survey of the different instabilities displayed by protein pharmaceuticals,
`by addressing which of these instabilities can be improved by glycosylation, and by
`discussing the possible mechanisms by which glycans induce these stabilization
`effects. ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci
`98:1223–1245, 2009
`Keywords: biopharmaceutics; biophysical models; chemical stability; glycosylation;
`molecular modeling; physical stability; physicochemical properties; proteins; stabiliza-
`tion; thermodynamics
`
`INTRODUCTION
`
`The employment of proteins as pharmaceutical
`agents has greatly expanded the field of molecular
`pharmacology as these generally display thera-
`peutically favorable properties, such as, higher
`target specificity and pharmacological potency
`
`Correspondence to: Ricardo J. Sola´ and Kai Griebenow
`(Telephone: 787-764-0000 ext 2391/4781; Fax: 787-756-8242;
`E-mail: rsola@bluebottle.com; kai.griebenow@gmail.com)
`
`Journal of Pharmaceutical Sciences, Vol. 98, 1223–1245 (2009)
`ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association
`
`when compared to traditional small molecule
`drugs.1,2 Unfortunately, the structural instability
`issues generally displayed by this class of
`molecules still remain one of the biggest chal-
`lenges to their pharmaceutical employment, as
`these can negatively impact their final therapeu-
`tic efficacies (Tab. 1).2–50 In contrast to traditional
`small molecule drugs whose physicochemical
`properties and structural stabilities are often
`much simpler to predict and control, the struc-
`tural complexity and diversity arising due to the
`macromolecular nature of proteins has hampered
`the development of predictive methods and
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 4, APRIL 2009
`
`1223
`
`Exhibit 2074
`Page 01 of 23
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`

`

`mStabilizationismostlyachievedbykeepingtheproteinawayfromdenaturinginterfacesorbysimplyavoidingsuchinterfacesaltogether.39,42,46,76
`lPrecipitationpriortotheprocedureaffordedstabilization.
`kThemechanismofstabilizationisbelievedtobeacombinationofhydrogen-bondformingpropensityandincreaseintheglasstransitiontemperatureinthesolid.23
`jSuchexcipientsincludesugars,polyols,andaminoacidsthatstabilizeproteinstructurebyso-calledpreferentialexclusion.2,75
`iMilddetergentsatlowconcentrationcanpreventdetrimentalinteractionsofproteinswithhydrophobicsurfaces/interfaces.42
`hToremovemetalions.2
`gOtherprominentchemicalinstabilitiesareoxidationsanddisulfidescrambling.2
`fAprominentpathwaytoaggregationisbyso-calledsulfide–disulfideinterchange.11
`eThepotentiallymostharmfulsurfacesarehydrophobic,e.g.,Teflon.45
`dControloftemperaturecanbenontrivialwhenultrasonicationisbeingusedbecauseoflocalheatingevents.
`cContaminating(transition)metalionsandproteasescancatalyzefragmentations.22
`bThesoleFDAapprovedformulationthusfarconsistsintheencapsulationoftheproteininmicrospherescomprisedofpoly(lactic-co-glycolic)acid.
`aThereferencescitedincludemanyreviewstowhichtheinterestedreaderisreferredtofordetails.
` CovalentmodificationascountermeasuresareexcludedinthetablebecausetheyarediscussedinthepaperandinTable2forglycosylatedproteins.
`
`1224
`
`SOLA´ AND GRIEBENOW
`
`39–44,77
`
`31–38,74
`
`4,16–18,30
`
`4,18,23–29,48,73
`
`50,68–72
`
`interfacesm
`avoidanceofwater/organic
`andstabilizingexcipients,j
`
`mechanicalstress
`hydrophobicsurfaces,e
`
`Additionofsurfaceactivei
`
`Aggregation,finactivation
`
`Liquid–organicsolventinterface,
`
`precipitationl
`
`formulationsb
`
`Sustained-release
`
`Spray-freezedrying
`
`Similartolyophilization,
`
`Similartolyophilization
`
`Liquid–airinterface,dehydration
`
`Spray-drying,
`
`Similartolyophilization
`stabilizingexcipientsj,k
`surfaceactiveiand
`Colyophilizationwith
`
`stabilizingexcipientsj
`surfaceactiveiand
`antioxidants,additionof
`chelatingagents,h
`
`inactivation
`oxidation,deamidation,
`
`contacts,moisturef
`
`Aggregation,ffragmentation,
`
`Contaminations,cprotein–protein
`
`Solid-phasestorage
`
`Aggregation,finactivation
`
`inactivation
`adsorption,aggregation,f
`deamidation,gdenaturation,
`b-elimination,racemization,
`crosslinking,
`hydrolysis,oxidation,
`
`dehydration,phaseseparation
`Ice–waterinterface,pHchanges,
`
`Lyophilization
`
`interfaces,hydrophobicsurfacese
`freezethawing,amphipatic
`highproteinconcentrations,
`temperature,dchemicaldenaturants,
`
`2,5–12,19–22,47,49,
`
`ControlofpHandtemperature,
`
`Fragmentations,chemical
`
`Contaminations,cextremesofpH,
`
`Liquidstorage
`
`2,5,6,10,19–22,68–72
`
`andstabilizingexcipientsj
`additionofsurfaceactivei
`agents,hantioxidants,
`temperature,chelating
`controlofpHand
`Proteaseinhibitors,
`
`inactivation
`adsorption,aggregation,f
`deamidation,gdenaturation,
`crosslinking,oxidation,
`hydrolysis,fragmentations,
`
`surfacese
`amphipaticinterfaces,hydrophobic
`highsaltandproteinconcentrations,
`temperature,dchemicaldenaturants,
`extremesofpH,highpressures,
`
`Proteolyticandchemical
`
`Proteases,contaminations,c
`
`Refs.a
`
`TypicalCountermeasures
`
`MainDegradationPathways
`
`MainStressFactors
`
`Purification
`
`Process
`
`Table1.ChemicalandPhysicalInstabilitiesEncounteredbyProtein-BasedPharmaceuticalsandTypicalCountermeasures
`
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`EFFECTS OF GLYCOSYLATION ON PROTEIN STABILITY
`
`1225
`
`generalized strategies concerning their chemical
`as well as their physical stabilizations.51,52 While
`the protein primary structure is subject to the
`same chemical instability issues as traditional
`small molecule therapeutics (e.g., acid-base and
`redox chemistry, chemical fragmentation, etc.),
`the higher levels of protein structure (e.g.,
`secondary, tertiary) often necessary for therapeu-
`tic efficacy can also result in additional physical
`instability issues (e.g.,
`irreversible conforma-
`tional changes, local and global unfolding) due
`to their noncovalent nature.2,15,53–55 The innate
`propensity of proteins to undergo structural
`changes coupled with the fact that there is only
`a marginal difference in thermodynamic stability
`between their folded and unfolded states provides
`a significant hurdle for the long-term stabilization
`of protein pharmaceuticals. This is due to the fact
`that a thermodynamically stabilized protein could
`still inactivate kinetically even at the relatively
`low temperatures used during storage.2,53,55–59
`Additionally, as a result of their colloidal nature,
`proteins are prone to pH, temperature, and
`concentration dependant precipitation, surface
`adsorption, and nonnative supramolecular aggre-
`gation.11,14,20,47,60–65 These instability issues are
`further compounded by the fact that the various
`levels of protein structure can become perturbed
`differently depending on the physicochemical
`environment to which the protein is exposed.2
`This is of special relevance in a pharmaceutical
`production setting where proteins can be simul-
`taneously exposed to several destabilizing envir-
`onments during their production, purification,
`storage, and delivery (Tab. 1).
`Due to these stability problems much emphasis
`has been given to the development of strategies for
`the effective long-term stabilization of protein
`pharmaceuticals.2,4,11,61,66–77 These include exter-
`nal stabilization by influencing the properties of
`the surrounding solvent through the use of
`stabilizing excipients (e.g., amino acids, sugars,
`polyols) and internal stabilization by altering the
`structural characteristics of the protein through
`chemical modifications (e.g., mutations, glycosy-
`lation, pegylation).2,53,58 While many protein
`pharmaceuticals have been successfully formu-
`lated by employing stabilizing mutations, excipi-
`ents, and pegylation, their use can sometimes be
`problematic due to limitations, such as, predicting
`the stabilizing nature of amino acid substitutions,
`the occurrence of protein and excipient dependant
`nongeneralized stabilization effects, protein/
`excipient phase separation upon freezing, cross-
`
`reactions between some excipients and the multi-
`ple chemical functionalities present in proteins,
`acceleration of certain chemical (e.g., aspartate
`isomerization) and physical (e.g., aggregation)
`instabilities by some excipients (e.g., sorbitol,
`glycerol, sucrose), detection interferences caused
`by some sugar excipients during various protein
`analysis methods, and safety concerns regarding
`the long-term use of pegylated proteins in vivo
`due to possible PEG induced immunogenecity
`and chronic accumulation toxicity resulting
`from its reduced degradation and clearance
`rates.2,4,33,48,66,78–95
`Due to these limitations, there is still a need
`for further development of additional strategies of
`protein stabilization.2 Amongst
`the chemical
`modification methods, glycosylation represents
`one of the most promising approaches as it is
`generally perceived that through manipulation of
`key glycosylation parameters (e.g., glycosylation
`degree, glycan size and glycan structural compo-
`sition) the protein’s molecular stability could be
`engineered as desired.2,66,96–105 In this context, it
`is important to highlight the fact that glycosyla-
`tion has been reported to simultaneously stabilize
`a variety of proteins against almost all of the
`major physicochemical instabilities encountered
`during their pharmaceutical employment (Tab. 2),
`suggesting the generality of these effects.
`Even though a vast amount of studies have
`evidenced the fact that glycosylation can lead to
`enhanced molecular stabilities and therapeutic
`efficacies for protein pharmaceuticals (Tab. 3), an
`encompassing perspective on this subject is still
`missing due to the lack of a comprehensive review
`of the literature. The intent of this article is
`therefore to further the field of protein glycoengi-
`neering by increasing the general understanding of
`the mechanisms by which glycosylation improves
`the molecular stability of protein pharmaceuticals.
`This is achieved by presenting a survey of the
`different instabilities displayed by protein phar-
`maceuticals, by addressing which of these instabil-
`ities can be improved by glycosylation, and by
`discussing the possible mechanisms by which
`glycans induce these stabilization effects.
`
`PROTEIN GLYCOSYLATION
`
`Protein glycosylation is one of the most common
`structural modifications employed by biological
`systems to expand proteome diversity.106–108
`Evolutionarily,
`glycosylation
`is widespread
`found to occur in proteins through the main
`
`DOI 10.1002/jps
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`1226
`
`SOLA´ AND GRIEBENOW
`
`Table 2. Protein Instabilities Improved by Glycosylation
`
`Instability
`
`Proteolytic degradation
`Oxidation
`Chemical crosslinking
`pH denaturation
`Chemical denaturation
`Heating denaturation
`
`Freezing denaturation
`Precipitation
`Kinetic inactivation
`Aggregation
`
`Refs.
`
`96,121–141
`145
`97,146,149
`124,137,171–178
`136,164,171,172,181–185,187,188
`98,101–103,119,124,128,129,146,149,159,170,171,181,182,
`188–195,202,204,205
`201
`159–165
`101,103,136,146,186,212–218
`97,101,103,130,218,222
`
`eubacteria, and
`(archaea,
`life
`of
`domains
`eukarya).109,110 The prevalence of glycosylation
`is such that it has been estimated that 50% of all
`proteins are glycosylated.111 Functionally, glyco-
`sylation has been shown to influence a variety of
`critical biological processes at both the cellular
`(e.g., intracellular targeting) and protein levels
`(e.g., protein–protein binding, protein molecular
`stability).103 It should therefore not come as a
`surprise that a substantial fraction of the cur-
`rently approved protein pharmaceuticals need to
`be properly glycosylated to exhibit optimal ther-
`apeutic efficacy.100,112
`Structurally, glycosylation is highly complex
`due to the fact that there can be heterogeneity
`with respect to the site of glycan attachment
`(macroheterogeneity) and with respect to the
`glycan’s structure (microheterogeneity). Although
`many protein residues have been found to be
`glycosylated with a variety of glycans (for a
`detailed discussion see review by Sears and
`Wong), in humans the most prevalent glycosyla-
`tion sites occur at asparagine residues (N-linked
`glycosylation through Asn-X-Thr/Ser recognition
`sequence) and at serine or threonine residues
`(O-linked glycosylation) with the following mono-
`saccharides: fucose, galactose, mannose (Man),
`N-acetylglucosamine (GlcNAc), N-acetylgalacto-
`samine, and sialic acid (N-acetylneuraminic
`acid).109,113–115 Since all of the potential glycosy-
`lation sites are not simultaneously occupied this
`leads to the formation of glycoforms with differ-
`ences in the number of attached glycans. Further
`structural complexity can occur due to variability
`in
`the
`glycan’s monosaccharide
`sequence
`order, branching pattern, and length. In humans
`N-linked glycan structures are classified in three
`principal categories according to their monosac-
`
`charide content and structure: high mannose
`type (Man2-6Man3GlcNAc2), mixed type (GlcNAc2-
`Man3GlcNAc2), and hybrid type (Man3GlcNAc-
`Man3GlcNAc2).113 The terminal ends of these
`glycans are often further functionalized with
`chemically charged groups (e.g., phosphates,
`sulfates, carboxylic acids) in human glycopro-
`teins, leading to even greater structural diversity.
`These charged glycans most probably impact to
`some degree the overall stability of glycoproteins
`since they can alter their
`isoelectric point
`(pI).116,117 Some of these charged terminal glycans
`(e.g., sialic acid) have also been found to be critical
`in regulating the circulatory half-life of glycopro-
`teins. This has led to the development of
`glycosylation as a novel strategy to improve the
`therapeutic efficacies of protein pharmaceuticals
`by engineering their pharmacokinetic profiles (for
`a detailed discussion see the recent review by
`Sinclair and Elliot).100
`Due to the high degree of structural variability
`arising from physiological (natural) glycosylation,
`novel strategies are currently being pursued to
`create structurally homogeneous pharmaceutical
`glycoproteins with humanized glycosylation pat-
`terns.118 These include engineered glycoprotein
`expression systems (e.g., yeast, plant, and mam-
`malian cells) as well as enzymatic, chemical, and
`chemo-enzymatic in vitro glycosylation remodel-
`ing methods. Alternatively, to understand the
`mechanisms by which glycosylation influences
`protein physicochemical properties researchers
`have employed comparatively simpler glycosyla-
`tion strategies. These include enzymatic deglyco-
`sylation of natural glycoproteins,
`chemical
`glycosylation via the use of structurally simple
`chemically activated glycans, and glycation of the
`lysine residues with reducing sugars via the
`
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`EFFECTS OF GLYCOSYLATION ON PROTEIN STABILITY
`
`1227
`
`(Continued)
`
`132
`
`149,159,160
`
`97
`
`142,194,195
`
`145,171,216,221
`
`127
`
`191
`
`101–103,188
`
`126
`
`181
`
`193
`
`161
`
`4
`
`3
`
`2
`
`b
`
`1
`
`2
`
`10
`
`b
`
`11
`
`3
`
`6
`
`3
`
`proteolyticdegradation
`
`Protectsagainst
`
`aggregation
`thermaldenaturation,and
`crosslinking,precipitation,
`
`Protectsagainstdisulfide
`
`crosslinkingandaggregation
`
`Protectsagainstnondisulfide
`
`thermaldenaturation
`
`Protectsagainstproteolysisand
`
`andaggregation
`inactivation,
`denaturation,kinetic
`thermal,chemical,andpH
`
`Protectsagainstoxidation,
`
`degradation
`
`Protectsagainstproteolytic
`
`denaturation
`
`Protectsagainstthermal
`
`denaturationandaggregation
`chemical,andkinetic
`
`Protectsagainstthermal,
`
`degradation
`
`Protectsagainstproteolytic
`andthermaldenaturation
`
`Protectsagainstchemical
`
`denaturation
`
`Protectsagainstthermal
`
`andprecipitation
`
`Protectsagainstaggregation
`
`Refs.
`
`Glycan(#)
`
`EffectsofGlycosylation
`
`granulomatousdisease
`
`Actimmune1(Intermune)Treatmentofchronic
`
`Interferongamma-1b
`
`Treatmentofmultiplesclerosis
`
`(Pfizer/EMDSerono)
`
`Avonex1(Biogen),Rebif1
`
`Interferonbeta-1a(rHuInf-b1)
`
`Treatmentofdiabetes
`
`Multipleindications
`
`a
`
`a
`
`Insulin
`
`IgG-likeantibodies
`
`withchronicrenalfailure(CRF)
`
`Treatmentofanemiaassociated
`
`Biotech)
`
`Epogen1(Amgen),
`
`Procrit1(Ortho
`
`Epoetinalfa
`ProteinC)
`
`Treatmentofseveresepsis
`
`Xigris1(EliLilly)
`
`Drotrecoginalfa(CF-XIV,
`
`Gonal-F1(EMDSerono)Treatmentofinfertility
`
`Corifollitropinalfa(FSH)
`
`myeloma
`
`Nutraceuticals)
`
`Adjuncttherapyformultiple
`
`insufficiency
`relatedtoexocrinepancreatic
`
`Treatmentoflipidmalabsorption
`
`deficiencywithemphysema
`
`Treatmentofcongenitala1-AT
`
`WobeMugos1(Marlyn
`
`Therapeutics)
`
`Merispase1(Meristem
`
`Biotherapeutics)
`
`Prolastin1(Talecris
`
`Chymotrypsin
`
`Bucelipasealfa(cholesterol
`
`esterase)
`
`Alpha1-antitrypsin(a1-AT)
`
`TreatmentofPompedisease
`
`Myozyme1(Shire)
`
`Alglucosidasealfa(a-glucosidase)
`
`TreatmentofFabrydisease
`
`Replagal1(Shire)
`
`Agalsidasealfa(galactosidase)
`
`Indication
`
`BrandName(Company)
`
`INN
`
`Table3.PartialListofApprovedProtein-BasedPharmaceuticalProductsStabilizedbyGlycosylation
`
`DOI 10.1002/jps
`
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`1228
`
`SOLA´ AND GRIEBENOW
`
`131,192
`
`130
`
`124,125,146,170
`
`128,129,189,190
`
`b
`
`8
`
`3
`
`2
`
`124,125,146,170
`
`1
`
`denaturation
`degradationandthermal
`Protectsagainstproteolytic
`
`degradationandaggregation
`
`Protectsagainstproteolytic
`
`andkineticinactivation
`thermalandpHdenaturation,
`degradation,
`crosslinking,proteolytic
`Protectsagainstdisulfide
`
`denaturation
`degradationandthermal
`Protectsagainstproteolytic
`
`kineticinactivation
`pHdenaturation,and
`degradation,thermaland
`crosslinking,proteolytic
`Protectsagainstdisulfide
`
`bCommerciallyavailableproteinisnotglycosylated.
`aMultipleapprovedproducts.Furtherinformationavailableatwww.fda.govandwww.biopharma.com.
`INN,Internationalnonproprietaryname.
`InformationwasobtainedfromthePrescribingInformation(PI)foreachproduct.
`
`pulmonaryemboli
`
`Therapeutics)
`
`Treatmentofacutemassive
`
`andhypothyroidism
`
`Abbokinase1(ImaRx
`
`Urokinasealfa
`
`Thyrogen1(Genzyme)Detectionofthyroidcancer
`
`Thyrotropinalfa(TSH)
`
`myelogenusleukemia
`chemotherapywithacute
`Leukin1(BayerHealthcare)Treatmentafterinduction
`
`Sargramostin(G-CSF)
`
`mesothelioma
`
`Onconase1(AlfacellCorp.)Treatmentofmalignant
`
`Ranpirnase(RNAse)
`
`inducedneutropenia
`
`(ChugaiPharma)
`
`Treatmentofchemotherapy
`
`Granocyte1
`
`Lenograstim(G-CSF)
`
`Refs.
`
`Glycan(#)
`
`EffectsofGlycosylation
`
`Indication
`
`BrandName(Company)
`
`INN
`
`Table3.(Continued)
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 4, APRIL 2009
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`EFFECTS OF GLYCOSYLATION ON PROTEIN STABILITY
`
`1229
`
`Maillard reaction. Although some of these glyco-
`sylation methods (e.g., glycation) may be unde-
`sired for use in protein pharmaceuticals their
`fundamental scientific value for the understand-
`ing the effects of glycosylation on protein stability
`cannot be ignored.119 This is due to the fact
`that independently of the method by which the
`structurally different glycans are attached to the
`protein surface (e.g., enzymatic and chemical
`glycosylation, or reductive glycation) they all
`seem to induce similar stabilization effects.103
`In the next sections, we thus focus on discussing
`which pharmaceutically relevant chemical and
`physical protein instabilities have been reported
`to be ameliorated by glycosylation and discuss
`possible mechanisms by which glycans achieve
`such effects.
`
`CHEMICAL INSTABILITIES PREVENTED BY
`GLYCOSYLATION
`
`The presence of multiple reactive chemical
`functionalities in the amino acids side chains of
`proteins makes them particularly sensitive to
`several chemical degradation processes. These
`can include: glutamine (Gln) and asparagine
`(Asn) deamidation; histidine (His), methionine
`(Met), cysteine (Cys),
`tryptophan (Trp), and
`tyrosine (Tyr) oxidation; serine (Ser), threonine
`(Thr), phenylalanine (Phe), lysine (Lys), and Cys
`b-elimination; disulfide fragmentation, exchange,
`and crosslinking; backbone peptide hydrolysis
`caused either by proteases or by pH sensitive
`backbone sequences (e.g., aspartic acid-proline
`(Asp-X));
`transamidation;
`racemization; and
`chemically triggered nonspecific
`crosslinking
`(Tab. 1).2,6,8,15,54,55,112 For further detailed dis-
`cussions on the general mechanisms which
`trigger these chemical instabilities the reader is
`referred to several excellent reviews on the
`subject.2,6,8,9,12,55 In the next section, we focus
`on those chemical instabilities which have been
`reported to be improved by glycosylation (e.g.,
`proteolytic degradation, oxidation, and chemical
`crosslinking) (Tab. 2).
`
`Proteolytic Degradation
`
`Protein pharmaceuticals are typically adminis-
`tered intravenously and not via the oral route due
`to their chemical degradation by the proteases of
`
`the digestive system.120 However, the systemic
`expression of proteases also makes proteins
`administered by other routes highly susceptibly
`to proteolytic degradation.120 Therefore,
`the
`in vivo molecular stability and therapeutic
`efficacy of protein pharmaceuticals is intimately
`related to their stability towards proteolytic
`degradation.2,6,100,120 In general, glycosylation
`has been found to protect proteins against
`proteolytic degradation.96,121–123 Some examples
`include granulocyte colony stimulating factor
`(GRANOCYTE1, Chugai Pharma,
`(G-CSF)
`(MERISPASE1;
`Tokyo, Japan),124,125
`lipase
`Meristem Therapeutics,
`Clermont-Ferrand,
`France),126 protein C (XIGRIS1; Eli Lilly,
`(ONCONASE1; Alfacell
`IN),127
`ribonuclease
`Corp., NJ),128,129 thyroid-stimulating hormone
`(THYROGEN1; Genzyme, MA),130 urokinase
`(ABBOKINASE1; ImaRx Therapeutics, AZ),131
`interferon-g (ACTIMMUNE1; Intermune, CA),132
`streptokinase,133 cellulose,134 ovomucoid,135 amy-
`lase,136,137 lysosomal integral membrane proteins
`Lamp-1 and Lamp-2,138 peroxidase,139 and cata-
`lase.140 There is also evidence that this proteolytic
`stability can be engineered into proteins as was
`described by Holcenberg et al.141 upon chemical
`glycosylation of asparaginase and by Raju and
`Scallon142 upon enzymatic glycosylation of IgG-
`like antibodies. Particularly, in this last study it
`was found that altering the end-terminal glycan
`structures (e.g., N-acetylglucosamine, galactose,
`and sialic acid) led to increasingly greater in vitro
`proteolytic stability when subjected to papain
`digestion.142 Mechanistically,
`it has been pro-
`posed that this proteolytic stability arises due to
`the fact that the glycan’s presence provides a
`steric hindrance around the peptide backbone of
`the amino acids adjacent to the glycosylation
`site.114,115,143 This prevents the contact between
`the glycoprotein’s surface and the cleaving pro-
`tease’s active site.
`
`Oxidation
`
`Protein pharmaceuticals can potentially lose their
`bioactivity during their manufacture and storage
`due to the oxidation of several of their amino
`acid side chains (His, Met, Cys, Trp, and
`Tyr).2,6,9,22,55,144 These oxidation events have
`been mainly attributed to the production of active
`oxygen-based radicals in protein formulations due
`to the combination of trace amounts of transition
`metals, atmospheric oxygen, and exposure to
`
`DOI 10.1002/jps
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`1230
`
`SOLA´ AND GRIEBENOW
`
`ultraviolet light.2,6 Thus far, erythropoietin (EPO-
`GEN1, PROCIT1; Amgen, CA, Ortho Biotech,
`NJ) is the sole reported case of a protein whose
`bioactivity can be impacted by oxidation and
`where glycosylation has been found to ameliorate
`this chemical instability.145 The loss of bioactivity
`for this protein was found to correlate with the
`levels of tryptophan oxidation when exposed to
`oxidizing conditions.145 Comparison of the oxida-
`tive susceptibility for the naturally glycosylated
`erythropoietin with that of its deglycosylated form
`revealed that glycosylation diminished the tryp-
`tophan oxidation rates and the inactivation of
`this protein.145 These results suggest that glycosy-
`lation can protect the protein structure from
`damage by active oxygen radicals although more
`studies are still needed to shed some light on the
`mechanisms of this stabilization and to determine
`the extent to which engineered glycosylation could
`prevent this type of instability. Also, whether this
`stabilizing effect is specific to when the glycans
`are chemically attached to the protein surface or
`nonspecific having to do more with the radical
`scavenging capabilities of the glycans remains to
`be established.70
`
`Chemical Crosslinking
`
`Protein therapeutics can form covalent dimers
`and oligomers due to polymerization triggered by
`both disulfide and nondisulfide crosslinking path-
`ways.2,6 Preventing the formation of these cova-
`lently linked species in protein pharmaceuticals is
`important as these frequently lead to loss of
`bioactivity.2,6 Additionally, for many proteins it
`has been found that this type of instability, in
`addition to protein unfolding, could trigger the
`formation of larger soluble and insoluble protein
`aggregates.2,6,11 There are several reports in the
`literature were it has been found that glycosyla-
`tion prevents the formation of these crosslinked
`species. For example, Oh-eda et al.146 reported
`that the presence of the single glycan in human
`granulocyte colony-stimulating factor (G-CSF)
`(GRANOCYTE1; Chugai Pharma) prevented
`the polymerization-induced inactivation of the
`protein. The mechanism by which G-CSF poly-
`merizes was studied by Krishnan et al. and Raso
`et al. and found to be due to disulfide cross-
`linking.147,148 Interferon beta (REBIF1, Pfizer,
`NY/Serono, Geneva, Switzerland; AVONEX1,
`Biogen, MA) is another example of a therapeu-
`tically relevant protein where glycosylation
`
`prevents its inactivation due to disulfide cross-
`linking.149 Glycosylation has been also reported to
`prevent nondisulfide protein crosslinking. For
`example, Baudys et al.97 reported that engineered
`chemical glycosylation of insulin, especially at the
`PheB-1 amino group, suppressed the self-associa-
`tion of the protein into dimers and oligomeric
`species. The formation of these crosslinked insulin
`species occurs due to a transamidation reaction
`between AsnA-21 and PheB-1.2 This finding is
`highly significant since it demonstrates that this
`type of stabilization can also be engineered into
`proteins via rationally designed glycosylation.
`These results additionally suggest
`that
`the
`mechanism by which this type of instability is
`prevented is due to increased intermolecular
`steric repulsion between the crosslinking-prone
`protein species due to the glycan’s presence at the
`protein surface.
`
`PHYSICAL INSTABILITIES PREVENTED
`BY GLYCOSYLATION
`
`The functional efficacy of proteins critically
`depends on the conformational stability of their
`natively folded state.2 Most proteins adopt a
`tertiary structure by folding as to minimize the
`exposure of their hydrophobic residues in aqueous
`solution.56,150–152 This creates a compact native
`state with a hydrophobic core that is additionally
`energetically stabilized by the presence of several
`types
`of
`atomic
`interactions within
`the
`protein core (e.g., electrostatic and charge–charge
`interactions, hydrogen bonds, Van der Waals
`interactions).151–154 Unfortunately, the resulting
`thermodynamic and kinetic stability of this state
`tends to be intrinsically low due to the noncova-
`lent nature of these forces.2,53 Therefore, any
`physical or chemical phenomena which can
`disrupt these forces will trigger either small or
`large scale protein structural changes. These
`conformationally altered species are more prone
`to interact either with themselves or with the
`hydrophobic surfaces and interfaces present
`during protein manufacturing and storage lead-
`ing to additionally physical instabilities, such as,
`adsorption, aggregation, and precipitation.2,42
`Examples of pharmaceutically relevant phenom-
`ena that can lead to protein physical instability
`include exposure to extremes of temperature and
`pH; exposure to amphipatic interfaces (e.g.,
`aqueous/organic solvent, aqueous/air), hydropho-
`bic surfaces, and chemical denaturants; and
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 4, APRIL 2009
`
`DOI 10.1002/jps
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`EFFECTS OF GLYCOSYLATION ON PROTEIN STABILITY
`
`1231
`
`formulation at extreme protein concentrations
`(Tab. 1). For further detailed discussions on the
`general mechanisms which trigger these physical
`instabilities the reader is again referred to
`a series of excellent
`reviews on the sub-
`ject.2,6,8,9,11,12,14,42,61,63,64 In the next section, we
`focus on those physical protein instabilities which
`have been reported to be improved by glycosyla-
`tion (e.g., precipitation; pH, chemical, and ther-
`mal denaturation; and aggregation) (Tab. 2).
`
`increased linearly as the glycosylation degree was
`increased (Fig. 2A).103,166 The linear dependence
`of these results are agreement with the solubility
`findings of Tams et al.164 These results therefore
`suggest the mechanism by which glycosylation
`increases protein solubility is due to an increase in
`the number of possible interactions between the
`glycoprotein surface and the surrounding solvent
`molecules due to an overall greater molecular
`SASA caused by the presence of the glycans.
`
`Precipitation
`
`One of the most fundamental challenges when
`designing a protein-based formulation involves
`achieving the desired therapeutic protein concen-
`tration in solution.2,63 This is due to the fact that
`protein solubility is not only inversely propor-
`tional
`to the protein concentration but also
`dependant on the solution’s pH, temperature,
`ionic
`strength,
`and
`excipient
`concentra-
`tion.2,52,63,155,156 Therefore, as the target concen-
`tration of the formulation is increased (e.g.,
`100 mg/mL) protein precipitation becomes a
`more critical problem.63 Glycosylation has been
`shown to increase the solubility of many pro-
`teins,99,157 although the generality of this effect
`has been questioned.158 Some examples include
`interferon beta (REBIF1, Pfizer/Serono; AVO-
`NEX1, Biogen),159,160 alpha-galactosidase A
`(REPLAGAL1, Shire, England, UK),161 glucose
`oxidase,162 and invertase.163 While studying the
`effects of glycosylation on peroxidase, Tams
`et al.164 determined that the solubility of the
`protein showed a linear dependence with the
`glycosylation degree. Although one could logically
`consider that this increased solubility is due to a
`greater hydration potential since the glycans have
`a higher affinity for the aqueous solvent than the
`polypeptide chain, Bagger et al.165 recently
`showed that this is not the case. From this study
`it was concluded that it is unlikely that strength-
`ened interactions with the aqueous solvent are the
`mechanism for increased protein solubility due to
`glycosylation.165 An alternative explanation can
`be provided from a comparative in silico struc-
`tural and energetic analysis recently performed
`by Sola´ and Griebenow on a series of chemically
`glycosylated a-chymotrypsin conjugates with
`increasing levels of glycosylation (Fig. 1).103,166
`From these computer simulations it was found
`that the overall molecular solvent accessible
`surface area (SASA) for the whole glycoprotein
`
`pH Denaturation
`
`Exposure of proteins to extremes of pH can result
`in loss of structure by disruption of both internal
`electrostatic forces and charge–charge inter-
`actions.2 At extreme pH values, far from the
`isoelectric point (pI), the unfolding propensity
`of proteins increases as a result of electrostatic
`repulsions
`between
`similarly
`charged
`atoms.2,151,167,168 Additionally, the diminished
`capability of salt bridge formation b