Journal of pharm'
`v “"3, no. 4
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`Volume 98 Number-4 April 2009
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`
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`Juiy 2008 - 24 November 2008
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`Page 1 of 29
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`CSL EXHIBIT 1060
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`Page 1 of 29
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`CSL EXHIBIT 1060
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`Journal of Pharmaceutical Sciences
`
`)
`
`{it} A Publication of the American Pharmacists Association
`APIIA
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`i A Publication of the Board of Pharmaceutical
`,65 Sciences of the International Pharmaceutical
`av.
`”'"m'"
`Federation
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`9) sans
`American Association of
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`Association of Pharmaceutical Scientists
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`Page 2 of 29
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`Volume 98, Number 4, April 2009
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`Overlooking Subvisible Particles in Therapeutic Protein Products: Gaps That May
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`Compromise Product Quality
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`John F. Carpenter,* Theodore W. Randolph, Wim Jiskoot, Dean J.A. Crommelin,
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`C. Russell Middaugh, Gerhard Winter, Ying-Xin Fan, Susan Kirshner, Daniela Verthelyi,
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`Steven Kozlowski, Kathleen A. Clouse, Patrick G‘ Swann, Amy Rosenberg, and Ba Ry Cherney
`Published online 14 August 2008
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`Biowaiver Monographs for Immediate Release Solid Oral Dosage Forms: Diclofenac
`Sodium and Diclofenac Potassium
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`1 206
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`B. Chuasuwan, V. Binjesoh, JE. Polli, H, Zhang, G.L. Amidon, l-l.E. Junginger, K.K. Midha,
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`V.P. Shah, S. Stavchansky, J.B. Dressman, and DM. Barends*
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`Published onl/‘ne 27 August 2008
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`Passing the Civilization Test
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`Joseph T. Cunliffe Sr.
`Published online 24 November 2008
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`",7 7 ,_#¥!
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`Effects of Glycosylation on the Stability of Protein Pharmaceuticals
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`Ricardo J. Sola* and Kai Griebenow
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`Published on/lne 25 July 2008
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`1 220
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`Principles, Approaches, and Challenges for Predicting Protein Aggregation Rates and
`Shelf Life
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`1 246
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`William F. Weiss lV, Teresa M.Young. and Christopher J. Roberts*
`Published online 6 August 2008
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`Volume 98, Number 4 was mailed the week of March 23, 2009,
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`In papers with more than one author, an asterisk (*) in the byline indicates the
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`author to whom inquiries should be directed
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`Page 3 0f 29
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`Vaccine Adjuvants: Current Challenges and Future Approaches
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`1 278
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`Jennifer H. Wilson-Welder, Maria P. Torres. Matt J. Kipper, Surya K. Mallapragada.
`Michael J. Wannemuehler, and Balaji Narasimhan'
`Published online 14 August 2008
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`From Natural Bone Grafts to Tissue Engineering Therapeutics: Brainstorming on
`Pharmaceutical Formulative Requirements and Challenges
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`1 31 7
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`Biancamaria Baroli
`Published online 26 August 2008
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`RESEARCH ARTICLES
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`l
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`BIOTECHNOLOGY
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`Structural Stability of Vault Particles
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`1 376
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`Reza Esfandiary. Valerie A. Kickhoefer, Leonard H. Rome. Sangeeia B. Joshi, and
`C. Russell Middaugh*
`Published online 6 August 2008
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`Solid State Chemistry of Proteins IV. What is the Meaning of Thermal Denaturation
`in Freeze Dried Proteins?
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`1387
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`Michael J. Pikal.* Daniel Rigsbee, and Michael J. Akers
`Published online 14 August 2008
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`Evaluation of the Effect of Non-B DNA Structures on Plasmid Integrity Via Accelerated
`Stability Studies
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`1 400
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`3.0. Ribeiro, G.A. Monteiro, and D.M.F. Prazeres'
`Published onllne 8 September 2008
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`DRUG DISCOVERY INTERFACE
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`Biochemical Mechanism of Acetaminophen (APAP) Induced Toxicity in Melanoma
`Cell Lines
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`1 409
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`Nikhil M. Vad, Garret Yount, Dan Moore, Jon Weidanz, and Majid Y. Moridani'
`Published online 29 August 2008
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`lPHARMACEUTICS, PREFORMULATION AND DRUG DELIVERY
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`NMR Search for Polymorphic Phase Transformations in Chlorpropamlde Form-A at High
`Pressures
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`1426
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`J. Wasickif D.P. Kozlenko, S.E. Pankov, P. Bilski, A. Pajzderska, 8.0. Hancock, A. Medek.
`W. Nawrocik. and 8N. Savenko
`Published online 11 July 2008
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`Synthesis, Characterization and In Vivo Activity of Salmon Calcitonin Coconjugated
`With Lipid and Polyethylene Glycol
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`1438
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`Weiqiang Cheng and Lee-Yong Lim‘
`Published online 14 August 2008
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`na- mnvarlnl a... Innicyi
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`Single and Double Emulsion Manufacturing Techniques of an Amphiphilic Drug in PLGA
`Nanopartlcles: Formulations of Mithramycin and Bioactivity
`Einat Cohen-Sela, Shay Teitlboim, Michael Chorny, Nickolay Koroukhov, Haim D. Danenberg,
`Jianchuan Gao, and Gershon Golomb*
`Published online 14 August 2008
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`Spray-Dried Carrier-Free Dry Powder Tobramycin Formulations With Improved
`Dispersion Properties
`
`Gabrielle Pilcer. Francis Vanderbist. and Karim Amighi‘
`Published online 27 August 2008
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`1452
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`1463
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`PHARMACEUTICAL TECHNOLOGY
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`Pair Distribution Function X-Ray Analysis Explains Dissolution Characteristics of
`Felodipine Melt Extrusion Products
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`1476
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`K. Nollenberger,* A. Gryczke, Ch. Meier, J. Dressman. M.U. Schmidt, and
`S. BriJhne
`Published online 27 August 2008
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`On-Line Monitoring of Pharmaceutical Production Processes Using Hidden
`Markov Model
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`1487
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`Hui Zhang,* Zhuangde Jiang, J.Y. Pi, H.K. Xu, and R. Du
`Published online 27 August 2008
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`Mapping Amorphous Material on a Partially Crystalline Surface: Nanothermal Analysis
`for Simultaneous Characterisation and Imaging of Lactose Compacts
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`1499
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`Xuan Dai, Mike Reading,’ and Duncan QM. Craig
`Published online 27 August 2008
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`Hydrodynamic, Mass Transfer, and Dissolution Effects Induced by Tablet Location during
`Dissolution Testing
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`1 51 1
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`Ge Bai and Piero M. Armenante"
`Published online 9 September 2008
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`PHARMACOKINETICS. PHARMACODYNAMICS AND DRUG METABOLISM
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`Physiological Models Are Good Tools to Predict Rat Bioavailability of EF5154 Prodrugs
`from In Vitro Intestinal Parameters
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`1532
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`Masahiro Nomotbf Tomoko Tatebayashi, Jun Morita, Hisashi Suzuki, Kazumasa Aizawa.
`Tohru Kurosawa, and Izumi Komiya
`Published online 6 August 2008
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`lnterindividual Pharmacokinetics Variability of the 044151 Integrin Antagonist,
`4-[1-[3-Chloro-4-[N'-(2-methylphenyl) ureido]phenylacetyl]-(4S)-fluoro-(ZS)-pyrrolidine-2-yl]
`methoxybenzoic Acid (D01-4S82), in Beagles Is Associated with Albumin Genetic
`Polymorphisms
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`1 545
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`Takashi Ito.‘ Masayuki Takahashi, Kenichi Sudo, and Yuichi Sugiyama
`Published online 14 August 2008
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`Pharmacokinetic and Pharmacodynamic Evaluation of Site-Specific PEGylated
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`Glucagon-Like Peptide-1 Analogs as Flexible Postprandial-Glucose Controllers
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`1556
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`Su Young Chae, Young Goo Chun, Seulki Lee. Cheng-Hao Jin, Eun Seong Lee, Kang Choon Lee, and
`Yu Seok Youn*
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`Published online 14 August 2008
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`Scintigraphic Study to Investigate the Effect of Food on a HPMC Modified Release
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`Formulation of UK-294,315
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`1563
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`J. Davis,* J. Burton, A.L. Connor, R. Macrae, and LR. Wilding
`Published online 27 August 2008
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`A Cremophor—Free Formulation for Tanespimycin (17-AAG) Using PEO-b-PDLLA Micelles:
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`Characterization and Pharmacokinetics in Rats
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`1577
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`May P. Xiong, Jaime A. Yafiez, Glen 8. Kwon, Neal M. Davies, and M. Laird Forrest*
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`Published online 27 August 2008
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`Pharmacokinetics of Amitriptyline and One of Its Metabolites, Nortript'yline. in Rats:
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`Little Contribution of Considerable Hepatic First-Pass Effect to Low Bloavailability of
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`Amitriptyline Due to Great Intestinal First-Pass Effect
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`1587
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`800 K. Bae, Kyung H.Yang, Dipendra K. Aryal, Yoon G. Kim, and Myung G. Lee*
`Published online 8 September 2008
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`REVIEWS
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`Effects of Glycosylation on the Stability of
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`Protein Pharmaceuticals
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`RICARDO ]. SOLA, KAI GRIEBENOW
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`Laboratory for Applied Biochemistry and Biotechnology, Department of Chemistry, University of Puerto Rico,
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`Rio Piedras Campus, Facundo Bueso Bldg, Lab«215, PO Box 23346, San Juan 00931—3346, Puerto Rico
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`Received 21 December 2007; revised 14 May 2008; acccpled 19 Junc 2008
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`Published online 25 July 2008 in Wiley InterSciencc (www.intersciencewiley.com). DOI 10.1002/jps.21504
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`ABSTRACT:
`In recent decades, protein-based therapeutics have substantially expanded
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`the field of molecular pharmacology due to their outstanding potential for the treatment
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`of disease. Unfortunately, protein pharmaceuticals display a series of intrinsic physical
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`and chemical instability problems during their production, purification, storage, and
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`delivery that can adversely impact their final therapeutic efficacies. This has prompted
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`an intense search for generalized strategies to engineer the long-term stability of
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`proteins during their pharmaceutical employment. Due to the well known effect that
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`glycans have in increasing the overall stability of glyeoproteins, rational manipulation of
`the glyeosylation parameters through glycoengineering could become a promising
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`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
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`increasing the general understanding of the mechanisms by which glycosylation
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`improves the molecular stability of protein pharmaceuticals. rI‘his is achieved by pre-
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`senting a survey of the different instabilities displayed by protein pharmaceuticals,
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`by addressing which of these instabilities can be improved by glycosylation, and by
`discussing the possible mechanisms by which glycans induce these stabilization
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`(9 2008 Wiley-Liss,
`Inc. and the American Pharmacists Association J Pharm Sci
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`981223—1245, 2009
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`Keywords: biopharmaceutics; biophysical models; chemical stability; glycosylation;
`molecular modeling; physical stability; physicochemical properties; proteins; stabiliza-
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`tion; thermodynamics
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`INTRODUCTION
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`The employment of proteins as pharmaceutical
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`agents has greatly expanded the field of molecular
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`pharmacology as these generally display thera-
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`peutically favorable properties, such as, higher
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`target specificity and pharmacological potency
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`when compared to traditional small molecule
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`drugsl‘2 Unfortunately, the structural instability
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`issues generally displayed by this class of
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`molecules still remain one of the biggest chal—
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`lenges to their pharmaceutical employment, as
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`these can negatively impact their final therapeu—
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`tic efficacies (Tab. 112—50 In contrast to traditional
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`small molecule drugs whose physicochemical
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`properties and structural stabilities are often
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`much simpler to predict and control, the struc-
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`tural complexity and diversity arising due to the
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`macromolecular nature of proteins has hampered
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`the development of predictive methods and
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`Correspondence to: Ricardo J. Sold and Kai Gricbenow
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`(Telephone: 787-764-0000 ext 2391/4781; Fax: 787-756-8242;
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`E-mail: rsolafitbluebottle.com; kaigriebenowftlgmail.c0m)
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`Journal of Pharmaceutical Sciences, Vol. 98, 1223—1245 (2009)
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`'9 2008 Wiley-Liss, Inc. and the American Pharmacists Association
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`~“““
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`IEZ’JYI‘ 59mm «
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`InterSCIencet
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`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APR” 2001)
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`Table 1. Chemical and Physical lnstabilities Encountered by Protein-Based Pharmaceuticals and Typical Countermeasures'
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`Main Stress Factors
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`Main Degradation Pathways
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`Refs."
`Typical Countermeasures
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`VZZI
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`MONElfli-llilf)GNVY'IOS
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`Proteolytic and chemical
`hydrolysis, frag-mentations,
`crosslinking, oxidation,
`deamidation,” denaturation,
`adsorption, aggregation,f
`inactivation
`
`Fragmentations, chemical
`hydrolysis, oxidation,
`crosslinking,
`B-elimination, racemization,
`deamidation,‘ denaturation,
`adsorption, aggregation,f
`inactivation
`
`Aggregation! inactivation
`
`Aggregation! fragmentation,
`oxidation, demnidation,
`inactivation
`Similar to lyophilization
`I‘
`
`Aggregation,
`
`inactivation
`
`Protease inhibitors,
`control of pH and
`temperature, chelating
`agents," antioxidants,
`addition of surface active"
`and stabilizing excipients’
`Control of pH and temperature,
`chelating agents."
`antioxidants, addition of
`surface activei and
`‘
`stabilizing excipients’
`
`Colyophilization with
`surface active" and
`stabilizing excipientsj‘l‘
`Similar to lyophilization
`
`Similar to lyophilization,
`precipitation,
`Addition of surface active"
`and stabilizing excipients,’
`avoidance of waterforganic
`interfaces“
`
`2,5,6,10,19—22,68—72
`
`2,5— 12,1922,1749
`50,68—72
`
`4,18,23—29,48,73
`
`4,16-18.30
`
`31—38,?4
`
`39—44.?7
`
`Process
`
`Purification
`
`Liquid storage
`
`Proteascs, contaminations,"
`extremes of pH, high pressures,
`temperature,d chemical denaturants,
`high salt and protein concentrations,
`amphipatic interfaces, hydrophobic
`surfaces”
`Contaminations,‘ extremes of pH,
`temperature,d chemical denaturants,
`high protein concentrations,
`freeze thawing, amphipatic
`interfaces, hydrophobic surfaces“
`
`Lyophilization
`
`Ice—water interface, pH changes,
`dehydration, phase separation
`
`Solid-phase storage
`
`Contaminations,‘ protein—protein
`contacts, moisturef
`
`Spray-drying,
`Spray-freeze drying
`Sustained-release
`formulationsb
`
`Liquid—air interface, dehydration
`
`Liquid-organic solvent interface,
`hydrophobic surfaces,”
`mechanical stress
`
`'Covalcnt modification as countermeasures are. excluded in the table because they are discussed in the paper and in Table 2 for glycosylated proteins.
`”The references cited include manv reviews to which the interested reader1s referred to for details.
`”The sole FDA apprmcd formulation thus far consists in the encapsulation of the protein in microspheres comprised of polytlacticvcoglycolicl acid.
`‘Contaminating (transition) metallens and proteases can catalvze frogmentations“
`“Control of temperature can be nontrivial when ultrasonicatiotiis being used because of local heating events.
`”1‘he potentially most harmful surfaces are hydrophobic e.g., Teflon.“
`[A prominent pathway to aggregation is by so-called sulfidMisulfide interchange“
`”Other prominent chemical instabilities are oxidations and disulfide scrambling."
`”To remove metalwas
`J'.‘
`'Mild detergents at low concentration can pre1ent detrimental interactions of proteins with h1drophobic surfaces}interfaces.
`'.'..5
`’Such excipients include sugars polvols, and amino acids that stabilize protein structure by so-called preferential cwlusion.
`The mechanism of stabilization1s believed to be a combination of hydrogen-bond forming propensity andincrease in the glass transition temperature in the solid.23
`39.42.~16.76
`'Precipitation 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.
`
`“ll/ZOOI'UI100
`
`Page 8 of 29
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`

`

`EFFECTS OF GLYCOSYLA’I‘ION ON PROTEIN STABILITY
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`1225
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`generalized strategies concerning their chemical
`
`
`
`
`
`
`
`as well as their physical stabilizationst52 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 (eg,
`
`
`
`
`
`secondary, tertiary) often necessary for therapeu-
`
`
`
`
`
`
`
`tic efflcacy can also result in additional physical
`
`
`
`
`instability issues (e.g.,
`irreversible conforma—
`
`
`
`
`
`
`
`tional changes,
`local and global unfolding) due
`
`to their noncovalent naturez’m’flfs‘i’ The innate
`
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`
`
`
`
`
`
`
`
`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.25’3’55’59
`
`
`
`
`
`
`
`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
`
`
`
`
`pharmaceuticalsg’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-
`
`
`
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`
`
`
`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-
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`
`
`DOI 10. l ()OZ/jps‘
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`Page 9 of 29
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`reactions between some excipients and the multi-
`
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`
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`
`
`ple chemical functionalities present in proteins,
`acceleration of certain chemical (e.g., aspartate
`
`
`
`
`
`
`
`
`
`
`
`isomerization) and physical (eg, aggregation)
`
`
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`
`
`
`instabilities by some excipients (e.g., sorbitol,
`
`
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`
`
`glycerol, sucrose), detection interferences caused
`
`
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`
`
`by some sugar excipients during various protein
`
`
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`
`
`analysis methods, and safety concerns regarding
`
`
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`
`
`the long-term use of pegylated proteins in vivo
`
`
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`
`
`due to possible PEG induced immunogenecity
`
`
`
`
`
`and chronic
`accumulation toxicity resulting
`
`
`
`
`
`
`from its
`reduced degradation and clearance
`
`
`mtesa4,33,4acmswoa
`
`
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`
`
`
`
`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 desiredg'GB'QG—mi’ 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 ’60
`enhanced molecular stabilities and therapeutic
`
`
`
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`
`
`
`
`
`
`
`
`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 ofprotein 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
`
`
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`
`
`discussing the possible mechanisms by WhiCh
`
`
`
`
`
`glycans induce these stabilization effects.
`
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`
`
`PROTEIN GLYCOSYLATION
`
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`
`
`Protein glycosylation is one of the most common
`
`
`
`
`
`structural modifications employed by biological
`
`
`
`to expand proteome diversity.lm"l08
`SYStems
`
`
`
`
`
`
`
`
`
`Evolutionarily,
`glycosylation
`is widespread
`
`
`
`
`
`
`
`found to occur in proteins through the main
`
`
`
`
`JOURNAL OF l’l’lARMACkUlICAL SCIENCES, VOL. 98, NO. 4, APRIL 2009
`
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`Page 9 of 29
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`

`

`1226
`
`
`
`SOLA AND GRIEBENOW
`
`
`
`
`
`
`
`Table 2. Protein lnstabilities Improved by Glycosylation
`
`
`
`
`
`
`
`
` Instability Refs.
`
`
`
`
`
`
`
`Protcolytic degradation
`
`Oxidation
`
`Chemical crosslinking
`
`pH denaturation
`
`
`Chemical denaturation
`
`Heating denaturation
`
`
`
`
`
`
`
`
`
`
`Freezing denaturation
`
`Precipitation
`
`Kinetic inactivation
`
`Aggregation
`
`
`
`
`
`
`
`96,121—141,
`145
`
`97,146,149
`
`124,137,171—178
`
`136,164,l71,172,181—185,187,188
`
`98,101—103,119,l,2<t,128,129,146,149,159,170,171,181,182,
`188—195,202,204,205
`
`201
`
`1597165
`
`
`101,103,136,146,186,212—218
`97,101,103,130,218,222
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`and
`of
`domains
`eubacteria,
`(archaea,
`life
`
`
`
`
`eul«:arya).1°9’110 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 01‘ 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).10(“l’“3‘115 Since all of the potential glycosy-
`
`
`
`
`
`
`
`
`
`
`
`
`
`latien 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
`
`
`
`
`sequence
`the
`glycan’s monosaccharide
`in
`
`
`
`
`
`
`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_(;Man;;GlcNAcz), mixed type (GICNACQ.
`
`
`
`
`MangGlcNAc2), and hybrid type (MangGlcNAC-
`
`
`
`
`
`MangGlcNAcig).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 glyeoproteins
`
`
`
`
`
`
`since they can alter
`isoelectric point
`their
`
`
`
`
`
`
`(p1). “6‘1” 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
`
`
`
`
`glycopreteins with humanized glycosylation pat-
`
`
`
`
`
`terns.118 These include engineered glycoprotein
`
`
`
`
`
`
`expression systems (eg, 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
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`JOURNAL OF PHARMACFUTICAL SCIENCES, VOL. 98, NO. 4, APRIL 2009
`
`
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`
`
`[)Ol 10.1002/jp5
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`
`Page 10 of 29
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`Page 10 of 29
`
`

`

`Stu/UK"inICU
`
`Table 3. Partial List of Approved Protein-Based Pharmaceutical Products Stabilized by Glycosylation
`INN
`Refs.
`
`Brand Name (Company)
`
`Indication
`
`Effects of Glycosylation
`
`Glycan (m
`
`161
`
`193
`
`181
`
`126
`
`101—103,188
`
`191
`
`127
`
`1']
`
`145,171.216,221
`
`142.194,]95
`
`97
`
`149,159,160
`
`132
`
`
`
`
`
`ALI’IIFWJSNIELLOlldN0NOI.].V'1:\SOOKID:10SJDSMJ
`
`(Contin uvcd l
`
`LZZI
`
`3 6 3 1
`
`1
`
`b
`
`10
`
`N)
`
`to
`
`Agalsidase alfa tgalactosidase)
`
`Replagal“ (Shirel
`
`Alglucosidase alfa (a-glucosidase)
`
`Myozyme” (Shire)
`
`Alpha l-antitrypsin («l-AT)
`
`Bucelipase alfa (cholester