`
`CSL EXHIBIT 1060
`
`Page 1 of 29
`
`CSL V. Shire
`
`Page 1 of 29
`
`CSL EXHIBIT 1060
`CSL v. Shire
`
`

`

`J ournal of Pharmaceutical Sciences
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`... ~ A Publication of the Am e ric a n Pharmacists Association
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`

`

`Volume 98, Number 4, April 2009
`
`J ournal of
`Pharmaceutical
`Sciences
`
`COMMENTARIES
`
`)
`
`Overlooking Subvisible Particles in Therapeutic Protein Products: Gaps That May
`Compromise Product Quality
`
`1201
`
`John F. Carpenter,* Th eodore W. Randolph, Wim Jiskoot, Daan J.A. Crommelin ,
`C. Russell Middaugh , Gerhard Winter, Ying-Xin Fan, Susan Kirshner, Daniela Verthelyi ,
`Steven Kozlowski , Kathleen A. Clouse, Patrick G. Swann, Amy Rosenberg , and Ba Ry Cherney
`Published online 14 August 2008
`
`Biowaiver Monographs for Immediate Release Solid Oral Dosage Forms: Diclofenac
`Sodium and Diclofenac Potassium
`B. Chuasuwan, V. Binjesoh, J.E. Polli , H. Zhang, G.L. Am idon, H.E. Junginger, K.K. Midha,
`V.P. Shah, S. Stavchansky, J.B. Dressman, and D.M. Barends*
`Published online 27 August 2008
`
`GLOBAL HEALTH COMMENTAR ~
`
`Passing the Civilization Test
`Joseph T. Cunliffe Sr.
`Published online 24 November 2008
`
`REVIEWS
`
`)
`
`1206
`
`1220
`
`Effects of Glycosylation on the Stability of Protein Pharmaceuticals
`
`1223
`
`Ricardo J. Sola* and Kai Griebenow
`Published online 25 July 2008
`
`Principles, Approaches, and Challenges for Predicting Protein Aggregation Rates and
`Shelf Life
`
`1246
`
`William F. Weiss IV, Teresa M. Young, and Christopher J. Roberts*
`Published online 6 August 2008
`
`Volume 98, Number 4 was mailed th e week of March 23, 2009.
`
`In papers with more than one author, an asterisk (*) in th e byline indicates the
`author to whom inquiries should be directed.
`
`Page 3 of 29
`
`

`

`Vaccine Adjuvants: Current Challenges and Future Approaches
`
`Jennifer H. Wilson-Welder, Maria P Torres, Matt J. Kipper, Surya K. Mallapragada,
`Michael J. Wannemuehler, and Balaji Narasimhan*
`Published online 14 August 2008
`
`1278
`
`From Natural Bone Grafts to Tissue Engineering Therapeutics: Brainstorming on
`Pharmaceutical Formulative Requirements and Challenges
`
`1317
`
`Biancamaria Baroli
`Published online 26 August 2008
`
`RESEARCH ARTICLES
`
`)
`
`Structural Stability of Vault Particles
`
`1376
`
`Reza Esfandiary, Valerie A. Kickhoefer, Leonard H. Rome, Sangeeta B. Joshi, and
`C. Russell Middaugh*
`Published online 6 August 2008
`
`Solid State Chemistry of Proteins IV. What is the Meaning of Thermal Denaturation
`in Freeze Dried Proteins?
`
`1387
`
`Michael J. Pikal,* Daniel Rigsbee, and Michael J. Akers
`Published online 14 August 2008
`
`Evaluation of the Effect of Non-B DNA Structures on Plasmid Integrity Via Accelerated
`Stability Studies
`
`1400
`
`S.C. Ribeiro, G.A. Monteiro, and D.M.F. Prazeres*
`Published online 8 September 2008
`
`Biochemical Mechanism of Acetaminophen (APAP) Induced Toxicity in Melanoma
`Cell Lines
`
`1409
`
`Nikhil M. Vad, Garret Yount, Dan Moore, Jon Weidanz, and Majid Y. Moridani*
`Published online 29 August 2008
`
`PHARMACEUTICS, PREFORMULATION AND DRUG DELIVERY
`
`•
`
`NMR Search for Polymorphic Phase Transformations in Chlorpropamide Form-A at High
`Pressures
`
`1426
`
`J. Wq_sicki,* D.P Kozlenko, S.E. Pankov, P Bilski, A. Pajzderska, B.C. Hancock, A. Medek,
`W. Nawrocik, and B.N. Savenko
`Published online 11 July 2008
`
`Synthesis, Characterization and In Vivo Activity of Salmon Calcitonin Coconjugated
`With Lipid and Polyethylene Glycol
`
`1438
`
`Weiqiang Cheng and Lee-Yang Lim*
`Published online 14 August 2008
`
`Page 4 of 29
`
`

`

`Single an? Double Emulsion Manufacturing Techniques of an Amphiphilic Drug in PLGA
`Nanopart1cles: Formulations of Mithramycin and Bioactivity
`
`Einat Cohen-Sela, Shay Teitlboim, Michael Chorny, Nicko lay Koroukhov, Haim D. Danenberg,
`Jianchuan Gao, and Gershon Golomb*
`Publi'l?&d online 14 August 2008
`
`Spray-Dried Carrier-Free Dry Powder Tobramycin Formulations With Improved
`Dispersion Properties
`
`Gabrielle Pilcer, Francis Vanderbist, and Karim Amighi *
`Published online 27 August 2008
`
`PHARMACEUTICAL TECHNOLOGY
`
`•
`
`-
`
`'"J
`
`-
`
`•
`
`•
`
`·
`
`I
`
`Pair Distribution Function X-Ray Analysis Explains Dissolution Characteristics of
`Felodipine Melt Extrusion Products
`
`K. Nollenberger,* A. Gryczke, Ch. Meier, J. Dressman , M.U. Schmidt, and
`S. Bruhne
`Published online 27 August 2008
`
`On-Line Monitoring of Pharmaceutical Production Processes Using Hidden
`Markov Model
`
`Hui Zhang ,* Zhuangde Jiang, J.Y. Pi, H.K. Xu, and R. Du
`Published online 27 August 2008
`
`1452
`
`1463
`
`1476
`
`1487
`
`Mapping Amorphous Material on a Partially Crystalline Surface: Nanothermal Analysis
`for Simultaneous Characterisation and Imaging of Lactose Compacts
`
`1499
`
`Xuan Da i, Mike Read ing, * and Duncan Q.M. Craig
`Published online 27 August 2008
`
`Hydrodynamic, Mass Transfer, and Dissolution Effects Induced by Tablet Location during
`D:ssolution Testing
`
`1511
`
`Ge Bai and Piero M. Armenante*
`Published online 9 September 2008
`
`Physiological Models Are Good Tools to Predict Rat Bioavailability of EF5154 Prodrugs
`from In Vitro Intestinal Parameters
`Masahiro Nomot~,* Tomoko Tatebayashi, Jun Morita, Hisashi Suzuki, Kazu masa Aizawa,
`Tohru Kurosawa, and Izumi Komiya
`Published online 6 August 2008
`
`1532
`
`lnterindividual Pharmacokinetics Variability of the ~4 131 lntegrin Antagonist,
`4-[1-[3-C h loro-4-[N '-(2-methyl phenyl) u rei do ]phenylacetyl]-( 4S)-fl uoro-(2S)-pyrrolidine-2-yl]
`methoxybenzoic Acid (D01-4582), in Beagles Is Associated with Albumin Genetic
`Polymorphisms
`
`1545
`
`Takash i Ito, * Masayuki Takahashi, Kenich i Sudo, and Yuichi Sug iyama
`Published online 14 August 2008
`
`Page 5 of 29
`
`

`

`Pharmacokinetic and Pharmacodynamic Evaluation of Site-Specific PEGylated
`Glucagon-Like Peptide-1 Analogs as Flexible Postprandial-Glucose Controllers
`
`1556
`Su Young Chae, Young Goo Chun, Seulki Lee, Cheng-Hao Jin, Eun Seong Lee, Kang Choon Lee, and
`Yu Seok Youn*
`Published online 14 August 2008
`
`Scintigraphic Study to Investigate the Effect of Food on a HPMC Modified Release
`Formulation of UK-294,315
`
`1568
`
`J. Davis,* J. Burton, A.L. Connor, R. Macrae, and I.R. Wilding
`Published online 27 August 2008
`
`A Cremophor-Free Formulation forTanespimycin (17-AAG) Using PEO-b-PDLLA Micelles:
`Characterization and Pharmacokinetics in Rats
`
`1577
`
`May P. Xiong, Jaime A. Yanez, GlenS. Kwon, Neal M. Davies, and M. Laird Forrest*
`Published online 27 August 2008
`
`Pharmacokinetics of Amitriptyline and One of Its Metabolites, Nortriptyline, in Rats:
`Little Contribution of Considerable Hepatic First-Pass Effect to Low Bioavailability of
`Amitriptyline Due to Great Intestinal First-Pass Effect
`
`1587
`
`Soo K. Bae, Kyung H. Yang, Dipendra K. Aryal, Yoon G. Kim, and Myung G. Lee*
`Published online 8 September 2008
`
`Page 6 of 29
`
`

`

`REVIEWS
`
`Effects of Glycosylation on the Stability of
`Protein Pharmaceuticals
`
`RICARDO ). SOlA, KAI GRIEBENOW
`
`Laboratory for Applied l3iochemistry and Biotechnology, Department of Chemistry, University of Puerto Rico,
`Rfo Piedras Campus, Facunclo 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). DOl 10.1002 /jps.21504
`
`In recent decades, protein-based therapeutics have substantially expanded
`ABSTRACT:
`the field of molecular pharmacol06'Y 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(cid:173)
`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. @ 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(cid:173)
`tion; thermodynamics
`
`INTRODUCTION
`
`The employment of proteins as pharmaceutical
`agents has greatly expanded the field of molecular
`pharmacology as these generally display thera(cid:173)
`peutically favorable properties, such as, higher
`target specificity and pharmacological potency
`
`Correspondence to: Hicardo J. Sola and Kai Griebenow
`(Telephone: 787-764-0000 ext 2391/t1781; Fax: 787-756-8242;
`E-mail: rsola((t,bluebottle.com; kai.griebenow(!,gmail.com)
`.Journal of Pharmaceutical Science,;, Vol. 98, 1223-1245 (2009)
`'') 2008 Wiley-Liss, Inc. and the American Pharmacists Association
`
`when compared to traditional small molecule
`drugs. 1•2 Unfortunately, the structural instability
`this class of
`issues generally displayed by
`molecules still remain one of the biggest chal(cid:173)
`lenges to their pharmaceutical employment, as
`these can negatively impact their final therapeu(cid:173)
`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(cid:173)
`tural complexity and diversity arising due to the
`macromolecular nature of proteins has hampered
`the development of predictive methods and
`
`'YJWILEY
`lnterScience''
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 4, APRIL 200'J
`
`1223
`
`Page 7 of 29
`
`

`

`Table 1. Chemical and Physical Instabilities Encountered by Protein-Based Pharmaceuticals and Typical Countermeasures'
`
`Main Stress Factors
`
`Main Degradation Pathways
`
`Typical Countermeasures
`
`Refs.a
`
`Process
`
`Purification
`
`Liquid storage
`
`Proteases, contaminations,'"
`extremes of pH, high pressures,
`temperature,d chemical denaturants,
`high salt and protein concentrations,
`amphipatic interfaces, hydrophobic
`surfacese
`Contaminations,c extremes of pH,
`temperature,d chemical denaturants,
`high protein concentrations,
`freeze thawing, amphipatic
`interfaces, hydrophobic surfacese
`
`Proteolytic and chemical
`hydrolysis, fragmentations,
`crosslinking, oxidation,
`deamidation,c denaturation,
`adsorption, aggregation/
`inactivation
`Fragmentations, chemical
`hydrolysis, oxidation,
`cross linking,
`[3-elimination, racemization,
`deamidation,c denaturation,
`adsorption, aggregation/
`inactivation
`Aggregation/ inactivation
`
`Aggregation/ fragmentation,
`oxidation, deamidation,
`inactivation
`Similar to lyophilization
`
`Aggregation/ inactivation
`
`Protease inhibitors,
`control of pH and
`temperature, chelating
`agents," antioxidants,
`addition of surface active;
`and stabilizing excipientsi
`Control of pH and temperature,
`chelating agents,"
`antioxidants, addition of
`surface active; and
`stabilizing excipientsi
`
`Colyophilization with
`surface active; and
`stabilizing excipientsj,l'
`Similar to lyophilization
`
`Similar to lyophilization,
`precipitation1
`Addition of surface active;
`and stabilizing excipients, 1
`avoidance of water/organic
`interfacesm
`
`2,5,6, 10,19-22,68-72
`
`2,5-12,19-22,4 7 ,49,
`50,68-72
`
`4,18,23-29,48,73
`
`4,16-18,30
`
`31-38,74
`
`39--44.77
`
`Lyophilization
`
`Ice-\vater interface, pH changes,
`dehydration, phase separation
`
`Solid-phase storage
`
`Contaminations, c protein-protein
`contacts, moisture""
`
`Spray-drying,
`Spray-freeze drying
`Sustained-release
`formulationsb
`
`Liquid-air interface, dehydration
`
`Liquid-organic solvent interface,
`hydrophobic surfaces:
`mechanical stress
`
`'Covalent modification as countermeasures are excluded in the table because they are discussed in the paper and in Table 2 for glycosylated proteins.
`0 The references cited include manv reviews to which the interested reader is referred to for details.
`bThe sole FDA approved formulation thus far consists in the encapsulation of the protein in microspheres comprised of poly(]actic-co-glycolicl acid.
`ccontaminating (transition) metal ions and proteases can catalyze fral,'1llentations. 2~
`dControl of temperature can be nontrivial when ultrasonication is being used because of local heating events.
`eThe potentially most harmful surfaces are hydrophobic, e.g., Teflon.45
`fA prominent pathway to aggregation is by so-called sulfide-disulfide interchange_ll
`gOther prominent chemical instabilities are oxidations and disulfide scrambling. 2
`"To remove metal ions. 2
`;Mild detergents at low concentration can prevent detrimental interactions of proteins with hydrophobic surfaces/interfaces.42
`75
`iSuch excipients include sugars, polyols, and amino acids that stabilize protein structure by so-called preferential exclusion. 2
`·
`kThe mechanism of stabilization is believed to be a combination of hydrogen-bond forming propensity and increase in the glass transition temperature in the solid. 23
`1Precipitation prior to the procedure afforded stabilization.
`"'Stabilization is mostly achieved by keeping the protein away from denaturing interfaces or by simply avoiding such interfaces altogether. 39
`
`2
`
`46
`•
`
`A
`
`6
`
`·'
`
`0
`Q
`
`0
`
`8
`%
`
`Page 8 of 29
`
`

`

`EFFECTS OF GLYCOSYLATION ON PHOTEIN 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(cid:173)
`tic efficacy can also result in additional physical
`instability issues (e.g.,
`irreversible conforma(cid:173)
`tional changes, local and global unfolding) due
`to their noncovalent nature. 2·1
`3-55 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-sn
`Additionally, as a result of their colloidal nature,
`proteins are prone to pH, temperature, and
`concentration dependant precipitation, surface
`adsorption, and nonnative supramolecular aggre(cid:173)
`gation.11·14·20.47,Go-Gs 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(cid:173)
`taneously exposed to several destabilizing envir(cid:173)
`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. 2,4,1l,Gl,GG-n 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(cid:173)
`lation, pegylation).2 •5:J,ss While many protein
`pharmaceuticals have been successfully formu(cid:173)
`lated by employing stabilizing mutations, excipi(cid:173)
`ents, and pef,>ylation, 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(cid:173)
`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,:l3,4S,GG,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(cid:173)
`sition) the protein's molecular stability could be
`engineered as desired. 2•6G·96- 105 In this context, it
`is important to highlight the fact that glycosyla(cid:173)
`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(cid:173)
`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(cid:173)
`maceuticals, by addressing which ofthese instabil(cid:173)
`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
`'t 106-108
`d .
`systems to expand proteome
`1vers1 y.
`is widespread
`Evolutionarily, glycosylation
`found to occur in proteins through the main
`
`001 10. I 002/jps
`
`JOURNAL OF I'Hi\RMACEUTICAL SCIENCES, VOL. <J8, NO. 4, APRIL 2009
`
`Page 9 of 29
`
`

`

`1226
`
`SOLA AND GRIEB EN OW
`
`Table 2. Protein Instabilities Improved by Glycosylation
`
`Instability
`
`Proteolytic degradation
`Oxidation
`Chemical crosslinking
`pH denaturation
`Chemical denaturation
`Heating denaturation
`
`Freezing denaturation
`Precipitation
`Kinetic inactivation
`Agf,'Tegation
`
`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
`
`(archaea, eubacteria, and
`life
`domains of
`eukarya). 109·110 The prevalence of glycosylation
`is such that it has been estimated that 50% of all
`proteins are glycosylated. 111 Functionally, glyco(cid:173)
`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(cid:173)
`rently approved protein pharmaceuticals need to
`be properly glycosylated to exhibit optimal ther(cid:173)
`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(cid:173)
`tion sites occur at asparagine residues (N-linked
`glycosylation through Asn-X-Thr/Ser recognition
`sequence) and at serine or threonine residues
`(0-linked glycosylation) with the following mono(cid:173)
`saccharides: fucose, galactose, mannose (Man),
`N-acetylglucosamine (GlcNAc), N-acetylgalacto(cid:173)
`samine, and sialic acid (N-acetylneuraminic
`.dl 109,113-115 s·
`.
`ac1
`.
`mce all of the potential glycosy-
`lation sites are not simultaneously occupied this
`leads to the formation of glycoforms with differ(cid:173)
`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 CMan2.GMan:lGlcNAc2), mixed type CGlcNAc2.
`Man3GlcNAc2), and hybrid type (Man3GlcNAc(cid:173)
`Man:3GlcNAc2).113 The terminal ends of these
`glycans are often further functionalized with
`chemically charged groups (e.g., phosphates,
`sulfates, carboxylic acids) in human glycopro(cid:173)
`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
`(p/). 116·117 Some of these charged terminal glycans
`(e.g., sialic acid) have also been found to be critical
`in ref,>Ulating the circulatory half-life of glycopro(cid:173)
`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(cid:173)
`terns. 118 These include engineered glycoprotein
`expression systems (e.g., yeast, plant, and mam(cid:173)
`malian cells) as well as enzymatic, chemical, and
`chemo-enzymatic in vitro glycosylation remodel(cid:173)
`ing methods. Alternatively, to understand the
`mechanisms by which glycosylation influences
`protein physicochemical properties researchers
`have employed comparatively simpler glycosyla(cid:173)
`tion strategies. These include enzymatic deglyco(cid:173)
`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
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. <Jil, NO. 4, Ar'RIL 2009
`
`DOl 1 0.1 002/jps
`
`Page 10 of 29
`
`

`

`Table 3. Partial List of Approved Protein-Based Pharmaceutical Products Stabilized by Glycosylation
`
`INN
`
`Brand Name (Company)
`
`Indication
`
`Effects of Glycosylation
`
`Glycan(#)
`
`Refs.
`
`Replagal 1' (Shire)
`
`Treatment of Fabry disease
`
`Myozyme" (Shire)
`
`Treatment of Pompe disease
`
`Protects against aggregation
`and precipitation
`Protects against thermal
`denaturation
`Protects against chemical
`Treatment of congenital nl-AT
`Prolastin " (Talecris
`and thermal denaturation
`deficiency with emphysema
`Biotherapeutics)
`Merispase " (Meristem Treatment of lipid malabsorption Protects against proteolytic
`Therapeutics)
`related to exocrine pancreatic
`degradation
`insufficiency
`Wobe Mugos" (Marlyn Adjunct therapy for multiple
`Nutraceuticals)
`myeloma
`
`Agalsidase alfa (galactosidase)
`
`Alglucosidase alfa (n-glucosidase)
`
`Alpha !-antitrypsin (nl-AT)
`
`Bucelipase alfa (cholesterol
`esterase)
`
`Chymotrypsin
`
`Corifollitropin alfa (FSH)
`
`Drotrecogin alfa (CF-XIV,
`Protein C)
`Epoetin alfa
`
`IgG-like antibodies
`
`Insulin
`
`0
`Q
`
`'" 0
`0 ""
`
`Gonal-F" (EMD Serono) Treatment of infertility
`
`Treatment of severe sepsis
`
`Xigris" (Eli Lilly)
`
`Epogen" (Amgen),
`Procrit" (Ortho
`Biotech)
`
`Multiple indications
`
`Treatment of diabetes
`
`Protects against thermaL
`chemical, and kinetic
`denaturation and aggregation
`Protects against thermal
`denaturation
`Protects against proteolytic
`degradation
`Treatment of anemia associated Protects against oxidation,
`with chronic renal failure (CRF)
`thermal, chemical, and pH
`denaturation, kinetic
`inactivation,
`and aggregation
`Protects against proteolysis and
`thermal denaturation
`Protects against nondisulfide
`crosslinking and aggregation
`Protects against disulfide
`crosslinking, precipitation,
`thermal denaturation, and
`aggregation
`Protects against
`proteolytic degradation
`
`Interferon beta-la (rHulnf-(31) Avonex" (Biogen), Rebif'' Treatment of multiple sclerosis
`(Pfizer/EMD Serono)
`
`Interferon gamma-lb
`
`Actimmune" (Intermune) Treatment of chronic
`granulomatous disease
`
`3
`
`6
`
`3
`
`11
`
`b
`
`10
`
`4
`
`3
`
`2
`
`b
`
`1
`
`2
`
`161
`
`193
`
`181
`
`126
`
`101-103,188
`
`191
`
`127
`
`145,171,216,221
`
`142.194,195
`
`97
`
`149,159,160
`
`132
`
`(Continued)
`
`Page 11 of 29
`
`

`

`Brand Name (Company)
`
`Indication
`
`Effects of Glycosylation
`
`Glycan(#)
`
`Refs.
`
`Granocyte"
`(Chugai Pharma)
`
`Treatment of chemotherapy
`induced neutropenia
`
`Protects against disulfide
`crosslinking, proteolytic
`degradation, thermal and
`pH denaturation, and
`kinetic inactivation
`Protects against proteolytic
`degradation and thermal
`denaturation
`Protects against disulfide
`crosslinking, proteolytic
`degradation,
`thermal and pH denaturation,
`and kinetic inactivation
`Protects against proteolytic
`degradation and aggregation
`Protects against proteolytic
`degradation and thermal
`denaturation
`
`1
`
`124,125,146,170
`
`b
`
`128,129,189,190
`
`8
`
`124,125,146,170
`
`3
`
`2
`
`130
`
`131,192
`
`Table 3. (Continued)
`
`INN
`
`Lenograstim (G-CSF)
`
`Ranpirnase (RNAse)
`
`Onconase" (Alfacell Corp.) Treatment of malignant
`mesothelioma
`
`Sargramostin (G-CSF)
`
`Leukin !' (Bayer Healthcare)Treatment after induction
`chemotherapy with acute
`myelogenus leukemia
`
`Thyrotropin alfa (TSH)
`
`Thyrogen" (Genzyme)
`
`Urokinase alfa
`
`Abbokinase " (!maRx
`Therapeutics)
`
`Detection of thyroid cancer
`and hypothyroidism
`Treatment of acute massive
`pulmonary emboli
`
`Information was obtained from the Prescribing Information (Pll for each product.
`INN, International nonproprietary name.
`aMultiple approved products. Further information available at www.fda.gov and www.biopharma.com.
`bCommercially available protein is not glycosylated.
`
`0
`
`Page 12 of 29
`
`

`

`EFFECTS OF GLYCOSYLATION ON PROTEIN STABILITY
`
`1229
`
`Maillard reaction. Although some of these glyco(cid:173)
`sylation methods (e.g., glycation) may be unde(cid:173)
`sired for use in protein pharmaceuticals their
`fundamental scientific value for the understand(cid:173)
`ing the effects of glycosylation on protein stability
`cannot be ignored. 119 This is due to the fact
`that independent