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`,VOIIImp 79 I Number RI Anmrcr 7fll I,
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`Pharmaceutical research.
`v. 28, no. 8 (Aug. 2011)
`General Collection
`W1 PH167H
`2011—08—24 10:39:42
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`@ aans An Official Journal of the American Assodation of Pharmaceutical Scientists
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`R E S E A R C H
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`"mm mmwm ov uum 1 @ Springer "
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`PROPERTY or THE
`NATIONAL
`LIBRARY OF
`MEQlClNE
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`”095 | ISSN 0724-874!
`28(8) l785-2058 (20! I)
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`This m-Mnriszl ”was Fania)!
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`CSL EXHIBIT 1067
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`Pharmaceutical Research
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`Volume 28 - Number 8 - August 2011
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`PERSPECTIVES
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`Finding Promiscuous Old Drugs for New Uses
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`S. Ekins ' AJ. Williams
`1785
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`EXPERT REVIEWS
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`Current and Future Drug Targets in Weight Management
`R.F. Witkamp
`1792
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`lntratumoral Drug Delivery With Nanoparticulatc Carriers
`H. Holback ' Y. Yeo
`1819
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`Effects of Surfactants on Lipase Structure, Activity
`and Inhibition
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`V. Delormc - R. Dhouib ~ S. Canaan ' F. Fotiadu -
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`F. Carriére - J.-F. Cavalier
`1831
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`Barriers to Non-Viral Vector-Mediated Gene Delivery
`in the Nervous System
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`PC. Perez-Martinez ‘ J. Guerra - l. Posadas ~ V, Cefia
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`1843
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`Effect of Formulation- and Administration—Related
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`Variables on Deposition Pattern of Nasal Spray Pumps
`Evaluated Using a Nasal Cast
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`V. Kundoor ~ RN. Dalby
`1895
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`Regioselective Glucuronidation of Flavonols by Six
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`Human UGT1A lsoforms
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`
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`B. Wu ~ B. Xu ' M. Hu
`1905
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`Laser-Engineered Dissolving Microneedle
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`Arrays for Transdermal Macromolecular
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`Drug Delivery
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`K. Migalska ' D.I.J. Morrow ' MJ. Garland - R. Thakur -
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`A.D. Woolfson - RF. Donnelly
`1919
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`Investigation of the Pharmacokinetics
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`of Romiplostim in Rodents with a Focus
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`on the Clearance Mechanism
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`Y.—M.C. Wang - B. Sloey ' T. Wong - P. Khandelwal -
`R. Melara - Y.—N. Sun
`1931
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`RESEARCH PAPERS
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`
`Validating New Tuberculosis Computational Models
`with Public Whole—Cell Screening Aerobic
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`Activity Datasets
`
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`S. Ekins - J.S. Frcundlich
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`
`1859
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`Enhanced Oral Bioavailability of Vinpocetine Through
`
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`Mechanochemical Salt Formation: Physico-chemical
`Characterization and In Vivo Studies
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`D. Hasa - D. Voinovich - B. Perissutti - M. Grassi -
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`A. Bonifacio - V. Sergo - C. Cepek - M.R. Chierotti -
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`R. Gobetto - S. Dall’Acqua ‘ S. lnvernizzi
`1870
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`Aggregation Stability of a Monoclonal Antibody
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`During Downstream Processing
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`P. Arosio ‘ G. Barolo ' T. Miiller—Spiith ' H. Wu '
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`M. Morbidelli
`1884
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`
`
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`
`
`
`
`Using Partial Area for Evaluation of Bioavailability
`and Bioequivalence
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`M.-L. Chen - B. Davit - R. Lionberger - Z. Wahba -
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`H.-Y. Ahn ~ L.X. Yu
`1939
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`A New Exact Test for the Evaluation of Population
`
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`Pharmacokinetic and/or Pharmacodynamic Models
`
`
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`Using Random Projections
`
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`CM. Lafibnt - D. Concordet
`
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`
`1948
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`Clinical Relevance of Liquid Chromatography Tandem
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`Mass Spectrometry as an Analytical Method
`in Microdose Clinical Studies
`
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`N. Yamane ' Z. Tozuka - M. Kusama ~ K. Maeda - T. lkeda -
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`Y. Sugiyama
`
`
`
`1963
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`Viscosity Analysis of High Concentration Bovine Serum
`Albumin Aqueous Solutions
`
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`S. Yadav - SJ. Shire - D.S. Kalonia
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`1973
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`Page 2 of 16
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`(E Springer
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`AAPS Connection
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`2055
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`Cover Image: from the article “Albumin-Coated Porous Hollow
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`Poly(Lactic-co-Glycolic Acid) Micropzitticles Bound with Palmityl—
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`Acylated Exendin—4 as a Long-Acting Inhalation Delivery System for
`
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`the ’l‘reatment of Diabetes,” pp. 2008—20 1 9
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`Further articles can be found at wwwspringerlinkcom.
`
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`Abstracted/lndexed in Hygiene and Communicable Diseases,
`
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`Academic Seam]: Alumni Edition, Academic Search Complete,
`
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`Academic Seam]! Premier BIOSIS Previews, Catalysts and
`
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`Catalysed Reactions, ClossFiIc Beilstein, CSA Biological Sciences,
`
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`
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`CSA Nemosciences Abstracts. Curie/1t Abstracts, Daily Science
`
`
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`
`
`
`Abstracts, EMBASE, Forest Pmclucts Abstracts, Global Health,
`
`
`
`
`
`
`
`Google Scholar Helmint/iological Abstracts, Horticultural Abstracts,
`
`
`
`
`
`
`IBIDS,
`Horticultural Science Abstracts,
`Index leterinarius,
`INIS
`
`
`
`
`
`
`
`
`
`
`
`
`Atomindex,
`INPHARMA,
`International I’lzannaccutical Abstracts,
`Journal Citation thorts/Social Sciences Edition, ll/Iass Specnometrv
`
`
`
`
`
`
`
`Bulletin, Nutrition Abstracts and Reviews Series A, OCLC ArticleFiIst
`
`
`
`
`
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`
`
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`
`
`Database, OCLC FirstSeaiclz Electronic Collections Online, BISCAL,
`
`
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`
`
`
`
`
`
`Pig News and liner/nation, Post/tarvest News and Information, Potam
`
`
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`Abstracts, Pmtazoological Abstracts, Pub/l/[erl/A/Ied/ine, Review of
`
`
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`
`
`
`Agricultural Entomology Review of Atomatic and tl/Iedicinal Plants;
`
`
`
`
`
`
`
`
`
`Review ofMedical and leterinmy Entomology, Review ofMetlical and
`
`
`
`
`
`
`
`
`leterinary Mycology Science Citation Index, Science Citation Index
`
`
`
`
`
`
`
`Expanded {SciSeatcb}, SCOPUS, Sugar Imlusny Abstracts, Summon
`
`
`
`
`
`
`
`by Serial Solutions, TOC Pzemier leterinary Bulletin.
`
`
`
`.
`lnstructions for authors for P/tarm Res are available
`
`
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`
`
`
`
`sprlngencom/IIOQS.
`
`
`
`
`'
`it
`
`1 WWW-
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`
`
`
`
`
`Poly(Lactide-c0—Glycolide) Nanocapsules Containing
`
`
`
`
`
`Benzocaine: Influence of the Composition
`
`
`
`
`
`
`of the Oily Nucleus on Physico—chemical Properties
`
`
`
`and Anesthetic Activity
`N.F.S. de Melo - R. Grillo ' V.A. Guilherme -
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`D.R. de Araujo - E. de Paula ~ A.H. Rosa -
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`L.F. Fraceto
`1984
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`How Stealthy are PEG-FLA Nanoparticles? An NIR
`In Vivo Study Combined with Detailed Size Measurements
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`A. Schéidlich - C. Rose ' J. Kuntsche ~ H. Caysa ‘
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`T. Mueller - A. Gopferich - K. Madcr
`1995
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`Albumin-Coated Porous Hollow Poly(Lactic-co-Glycolic
`
`
`
`Acid) Microparticles Bound with Palmityl-Acylated
`
`
`
`
`
`Exendin—4 as a Long-Acting Inhalation Delivery System
`
`
`
`
`
`
`for the Treatment of Diabetes
`
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`
`H. Kim - J. Lee - T.H. Kim - E.S. Lee - K.T. Oh - D.H. Lee -
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`E.-S. Park ' Y.H. Bae - K.C. Lee - Y.S. Youn
`2008
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`Formulation, Biological and [’harmacokinetic Studies
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`of Sucrose Ester-Stabilized Nanosuspensions
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`of Oleanolic Acid
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`W. Li ~ S. Das ~ K.—y. Ng ' P.W.S. Heng
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`
`
`2020
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`The Role of Constitutive Androstane Receptor
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`in Oxazaphosphorine-Mediated Induction
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`of Drug-Metabolizing Enzymes in Human IIepatocytes
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`D. Wang ‘ L. Li - J. Fuhrman - S. Ferguson -
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`H. Wang
`2034
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`Increased Liver Uptake and Reduced Hepatic Stellate
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`Cell Activation with a Cell-Specific Conjugate
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`of the Rho-kinase Inhibitor Y27632
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`M.M. van Beuge - J. Prakash ~ M. Lacombc - E. Post -
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`C. Rcker-Smit - L. Beljaars - K. Poelstra
`2045
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`{ASpfin‘ger
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`PHARMACEUTICAL RESEARCH
`An Official .Iouriial of the American Association of Pharmaceutical Scientists
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`l’lnrrnrnccu/icul Research, an officral journal of the American Association of Pharmaceutical Scientists, publishes papers on innovative research spanninw the entire spectrum of
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`science that is the loundation of drug discovery, development, evaluation, arid regulatory approval. Small drug molecules, biotechnology products incaludingy genes peptides
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`proteins and vaccrrtes,'and geneltcplly engineered cells are an integral part of papers published in Pharmaceutical Research. Current emphasis of the journal includes, but is noi
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`limited to, the following areas: lr'clormulatron; drug delivery and targeting; formulation design, engineering, and processing; plrarmacokinetics, pliarmacodynzirnics, and
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`pharmacogenonrics; molecular hiopharinaceutics and drug disposition; and computational biopharinaceutics
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`EDI'I‘OR-INCIIIEF
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`Peter W. Swarm, University of Maryland, Baltimore, Maryland, USA
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`EDITORS
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`Paril M. Bummer, University of Kentucky, Lexington, Kentucky, USA
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`Margareta IIammarlunrl-Urlenaes, Uppsala University, Uppsala, Sweden
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`Ken-ichi Inui, Kyoto Pharmac ulical University, Kyoto, Japan
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`Wim Jiskoot, Leiden University, Netherlands
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`Uday Kompclla, University of Colorado, Denver, Colorado, USA
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`All-Ng Tony Kong, State University of New Jersey—Rutgers, Piscataway,
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`New Jersey, USA
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`Tamara Minko, State University of NewJersey—Rutgers, Piscataway, NewJersey, USA
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`James E. Polli, University of Maryland, Baltimore, Maryland, USA
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`David E. Smith, University of Michigan, Ann Arbor, Michigan, USA
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`Christine Vaiitliier, Centre National de la Recherche Seicntifique, Paris, France
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`Ernst Wagner, Ludwig-Maximilians-Universitiit, Munich, Germany
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`Yuhong Xn, Shanghai Jiao Tong University Med-X Research Institute,
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`Shanghai, China
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`EDITOR, EXPERT REVIEWS
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`Sean Ekins, Collaborations in Chemistry, Philadelphia, Pennsylvania, USA
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`EDITOR, SPECIAL FEATURES
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`Rani I. Mahatn, University of Tennessee, Memphis, Tennessee, USA
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`CONSULTING EDITORS
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`Per Artnrsson, Uppsala University, Uppsala, Sweden
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`Jessie L-S. Au, Ohio State University, Columbus, Ohio, USA
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`Dam] .I.A. Crommelin, Utrecht Institute for Pharmaceutical Sciences,
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`Utrecht, Netherlands
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`Wiirr E. Ilennink, Utrecht Institute for Pharmaceutical Sciences,
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`Utrecht. Netherlands
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`William .Insko, SUNY Buffalo, Buffalo, New York, USA
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`Nicholas A. l’eppas, University of Texas at Austin, Austin, ’I‘exag, USA
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`Joseph W. Polli, GlaxoSmithKline, Research Triangle Park, North Carolina, USA
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`Wolfgang Sade’e, Ohio State Universrty, Columbus, Ohio, USA
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`Danny Sheri, University of Washington, Seattle, Washington, USA
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`Yuiclii Sngiyanra, University of Tokyo. Tokyo, Japan
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`Benign-d Tesla, University Hospital Center, Laussane, Switzerland
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`EDITORIAL ADVISORY BOARD
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`Sandra R.B. Allerhu tgen, Eli Lilly, Indianapolis, Indiana, USA
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`Bradley I). Anderson, University of Kentucky, Lexington, Kentucky, USA
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`Patrick Arigiistijiis, Katholieke Universiteit Leuven, Leuven, Belgium
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`You-Hun Bae, University of Utah, Salt Lake City, Utah, USA
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`Reina Bendayan, University of Toronto, Toronto, Canada
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`Andreas Bernkop-Schnr‘ircli, University of Innsbruck, Innsbruck, Austria
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`Robin II. Bogner, University of Connecticut, Storrs, Connecticut, USA
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`Kathleen M.K. Boje, SUNY Buffalo, Buffalo, New York, USA
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`Yoiingrt) Byun, Seoul National University, Seoul, Korea
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`John F. Carpenter, University of Colorado, Denver, Colorado, USA
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`Ilak Kim Chan, University of Sydney, Sydney, Australia
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`Albert ILL, Chow, Chinese University of llong Kong, Sha Tin, Hong Kong
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`Paolo Colomlio, University of I’arma, l’arina, Italy
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`James ’I‘. Dalton, Ohio State University, Columbus, Ohio, USA
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`Gérard Déléris, University of Bordeaux, Bordeaux, France
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`William F. Elmqiiist, University of Minnesota, Minneapolis, Minnesota, USA
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`Gert Frickur, RuprechtharlsAUiriversitat Heidelberg, Heidelberg, Germany
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`Lawrence Gait, Millennium Pharmaceuticals, lnc., Cambridge, Massachusetts, USA
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`Vadivel (lanapathy, Medical College of Georgia, Augusta, Georgia, USA
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`Bruno Gander, ETH Zfirieh Institute of Pharmaceutical Sciences,
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`Ilainidreza Ghanrleliari, University of Utah, Salt Lake City, Utah, USA
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`Scott Grossnian, BristoLMyers Squibb, Wallingford, Connecticut, USA
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`Hyo-Kyung Han, Dongguk University, Seoul, Korea
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`Bruno C. Hancock, Pfizer lnc., Groton, Connecticut, USA
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`llideyoshi Harasliima, Hokkaido University, Sapporo, Japan
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`Ian I'Iawortli, University of Southern California, Los Angeles, California, USA
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`Anthony J. Hickey, University of North Carolina at Chapel Hill, Chapel Hill,
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`North Carolina. USA
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`Kathleen "illgren, Eli Lilly, Indianapolis, Indiana, USA
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`Gunther lIoclihans, University of Florida, Gainesville, Florida, USA
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`Nicholas II.G. Ilolford, University of Auckland, Auckland, New Zealand
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`Kcn-ichi Hosoyn, Toyama University, Toyarna, Japan
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`.lin-I)ing Huang, National Cheng»Kung University, Tainan, Taiwan
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`Ajal. Ilussain, Philip Morris International, Washington, DC, USA
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`Toshihiko Ikcda, Sankyu Co. Ltd,, Tokyo, Japan
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`Alexander V. Kabanov, University of Nebraska, Omaha, Nebraska, USA
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`lliroyiiki Krisrihara, University of Tokyo, Tokyo, Japan
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`Glen S. Kwon, University of Wisconsin, Madison, Wisconsin, USA
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`Chills-Michael Lelrr, Saarland University, Saarbrueken, Germany
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`Tonglei Li, University of Kentucky, Lexington, Kentucky, USA
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`.Iiunn I-I. Lin, Merck Research Laboratories, Westpoint, Pennsylvania, USA
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`Dcxi Liu, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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`Zheng-Rong Ln, University of Utah, Salt Lake City, Utah, USA
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`Palltls Macheras, University of Athens, Athens, Greece
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`Patrick McNamara, University of Kentucky, Lexington, Kentucky, USA
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`David Mooney, Harvard University, Cambridge, Massachusetts, USA
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`Marilyn E. Morris, SUNY Buffalo, Buffalo, New York, USA
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`Eini Nakashimn, Kyroitsu University, Tokyo, Japan
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`Tae Gwan Park, Korea Advanced Institute of Science and 'I‘echnology,
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`Deajon, Korea
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`Chris Porter, Monash University, Melbourne, Australia
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`Mark I'rausnltz, Georgia Institute of Technology, Atlanta, Georgia, USA
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`David Putnam, Cornell University, Ithaca, New York, USA
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`Michael Roberts, University of Queensland, Brisbane, Australia
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`Patrizia Santi, University of Patina, Parma, Italy
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`Chang-Koo Shim, Seoul National University, Seoul, Korea
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`Steven J. Shire, Gciientech, lnc., San Francisco, California, USA
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`Alldra L. Stinchcomb, University of Kentucky, Lexington, Kentucky, USA
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`I'Iiroshi Suzuki, University of Tokyo Hospital, Tokyo, Japan
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`Yoshinolui Takaknra, Kyoto University, Kyoto, Japan
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`lknmi ’I‘amai, Kanazawa University, Kakuma, Japan
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`lfiaki It‘.
`'I‘roconiz, University of Navarra, Pamplona, Spain
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`Kislror M. Wasan, University of British Columbia, Vancouver, Canada
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`Ronald E. White, Sclieriiig—Plough Research Institute, Kenilworth,
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`New Jersey, USA
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`Guillaume Wientjes, Ohio State University, Columbus, Ohio, USA
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`Weir Xie, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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`Kciji Yamamoto, Chiba University, Chiba, Japan
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`FIIIIIIyOSIli Yamaslrita, Kyoto University, Kyoto, Japan
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`Gnol’cng You, State University of New Jersey—Rutgers, Piscataway,
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`EDITORIAL ASSISTANT
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`Rachel I). Lucke, University of Maryland, Baltimore, Maryland, USA
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`

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`Pharm Res (20I I) 28:|973—l983
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`DOl lO.|007/5| |095—0l [—0424—7
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`i RESEARCH PAPER
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`Viscosity Analysis of High Concentration Bovine Serum
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`Albumin Aqueous Solutions
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`Sandeep Yadav - Steven 1. Shire - Devendra S. Kalonia
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`Received: 8 December 2010 /Aeceptcd: 8 March 2011 /l’ublished online:
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`ii?) Springer Science+Business Media, LLU 2011
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`1/1: April 201 1
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`ABSTRACT
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`Purpose To understand the apparent inconsistency between
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`the dilute and high concentration viscosity behavior of bovine
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`serum albumin (BSA).
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`Method Zeta potential and molecular charge on BSA were
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`determined from Electrophoretic mobility measurements.
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`Second virial coefi‘icient (522) and interaction parameter (kD)
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`obtained from static and dynamic light scattering, respectively,
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`quantified intermolecular interactions. Rheology studies char—
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`acterized viscoelasticity at high concentration. The dipole
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`moment was calculated using Takashima’s approximation for
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`proton fluctuations over charged residues.
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`Results The effective isoelectric point of BSA was pH 4.95. In
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`dilute solutions (5 40 mg/ml), the viscosity was minimal at the
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`pl; at high concentrations. pH 5.0 solutions were most viscous.
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`522 and k0 showed intermolecular attractions at pH 5.0;
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`repulsions dominated at other pl—ls. The attractive interactions
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`led to a high storage modulus (G') at pH 5.0.
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`Electronic supplementary material The online version of this article
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`(doi: l O. l007/sl |O95-Ol 1—0424-7) contains supplementary material,
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`which is available to authorized users.
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`S. Yadav ' D. S. Kalonia (E)
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`Department of Pharmaceutical Sciences, University of Connecticut
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`Storrs, CT 06269, USA
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`e-mail: kalonia@uconn,edu
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`S. J. Shire
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`Late Stage Pharmaceutical Development
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`Genentech, Inc.
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`i DNA Way
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`South San Francisco, CA 94080, USA
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`Present Address:
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`S, Yadav
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`Late Stage Pharmaceutical Development
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`Genentcch, Inc.
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`South San Francisco, CA 94080. USA
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`Page 5 0f 16
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`Conclusion in dilute solutions, the electroviscous effect due to
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`net charge governs the viscosity behavior; at high concen4
`trations, the solution viscosity cannot be justified based on a
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`single parameter. The net interplay of all intermolecular forces
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`dictates viscosity behavior, wherein intermolecular attraction
`leads to a higher solution viscosity.
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`KEY WORDS dipole moment . high concentration viscosity-
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`interaction parameter (kD).
`- intermolecular interaction - protein
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`charge - second virial coefficient (Bu) . zeta potential
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`INTRODUCTION
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`In 1956 Buzzel and Tanford published the Viscosity 01‘
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`bovine serum albumin (BSA) and ril')onuclease (RNAase) at
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`various conditions of solution pH and ionic strengths (1,2).
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`For the concentration range studied (~40e50 mg/ ml) the
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`solution viscosity showed a good correlation with the net
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`charge-induced electroviscous effects. Due to the presence
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`of electrical charge on the molecule,
`three kinds ol‘
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`contribution may zillcct the viscosity behavior. A ‘primzuy
`ellcct’ due to the resistance oi~ the clillilse double layer
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`surrounding the molecule, a ‘secondary eflect’ due to the
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`intermolecular repulsion between double layers and a
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`‘tertiary ellect’ that may arise if the intcrparticle repulsion
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`allects the shape 01‘ the l'naeromolecule. These three are
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`collectively known as the ‘electroviscous eliccts’ (3). When a
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`charged particle moves through a medium comprising
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`small
`ions, electrostatic interaction between the particle
`and the small ions results in a relative motion of the ions to
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`the medium and consequently an additional viscous loss
`arises that contributes to the overall viscosity ol'tlre solution.
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`For BSA and RNAase solutions the slope of the reduced
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`viscosity (17M),
`i.e.
`the specific increment in viscosity as a
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`Page 5 of 16
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`

`

`I974
`Yadav. Shire and Kalonia
`
`increased with an
`function of protein concentration (6),
`increase in molecular charge (1,2). An increase in solution
`ionic strength resulted in a decrease in the slope ("ml yen-([5 c),
`which finally attained a limiting value at high solution ionic
`strengths. The authors attributed this to the net molecular
`charge—induced primary and secondary clectroviscous eifects,
`which also correlated to some extent with Booth’s theory (-1-)
`of anticipated increase in intrinsic viscosity [)7] due to
`electroviscous elfccts (1,2).
`The observed behavior suggests that the viscosity should
`increase with an increase in the molecular charge due to the
`additional resistance to flow offered by the electroviseous
`cllects.
`It
`then follows that for a protein solution the
`viscosity should be minimal at the isoeleetrie poirtt (pl),
`when the net molecular charge is zero, and should increase
`as the solution conditions are made more acidic or basic
`relative to the pl. For dilute protein solutions, this trend has
`generally been observed (5,6). Funlrennorc, for BSA solution,
`certain calculations have been presented using the original data
`in Tanlbrd’s work (2), which supports this argument (Fig. 1).
`The details of the calculation for Fig.
`l have been explained
`in the Discussion section of this work.
`
`Recent studies on the viscosity behavior of high
`concentration protein solutions have shown an altogether
`dilfcrent behavior. The viscosity for 120 mg/ ml
`lgGg
`solution was observed to be highest at the pl (7), which is
`not
`in agreement with the net charge-induced electro-
`viscous effects. Conversely, 130 mg/ ml MAb~l (lgGl), with
`a measured pl of 7.8, showed the highest viscosity at pH 6.0
`relative to other pHs studied (8,9). Salinas at (1!. suggested
`that
`the high viscosity observed for >50 mg/ml
`lgG.
`solution, at pH 6.0 was primarily due to electroviscous
`effects (10). On the contrary, Yadav et a1.
`(11) did not
`observe a consistent interpretation of electroviscous ell'ects
`to the viscosity behavior of four dilfcrent MAbs. (l l)
`The dilute solution viscosity behavior of BSA, wherein
`the viscosity was observed to be minimal at the pl (Fig.
`I),
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`tl'rerefore, does not correlate with the recently published
`high concentration viscosity data on lgG molecules. The
`present study seeks to understand the apparent inconsistency
`between the dilute and high concentration viscosity behavior
`of protein solutions. In particular, the high concentration
`viscosity behavior of BSA solutions as well as the dilfcrerrt
`factors that may be resprmsiblc for the observed behavior
`have been analyzed and discussed.
`
`MATERIALS AND METHODS
`
`Materials
`
`The BSA (lyoplrilized, purity 99% and essentially fatty acid-
`and globulin-free (Catalogue number: A0281) was obtained
`front Sigma (St. Louis, MO). All other chemicals including
`acetic acid, sodium acetate, sodium chloride, histidine
`hydrochloride, monohasic and dibasic sodium phosphate,
`were obtained from Fisher Scierrtilie (Fair lawn, NJ). All
`chemicals used were reagent grade or higher. Dcionizcd
`water equivalent to l‘vlilli-QL"M grade was used to prepare
`all solutions. Millipore (Billerica, MA) Amieon Ultra
`centrifugation tubes with a molecular weight cut-oll‘ of
`3 kD were obtained from Fisher Scientific. Quartz crystal
`discs with fundamental vibrating frequencies of IO MHz
`and plated with gold electrodes on both sides were
`obtained from International Crystal h'lantrfaeturing
`Company (Oklahoma City, Oklahoma).
`
`Methods
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`Acetic acid-sodium acetate (pH 4.0, 5.0), lristidine hydrochlo-
`ride {pl I (3.0) and rnorrobasie-dibasic sodium phosphate [pll 7.0
`and 8.0) bullers were prepared with appropriate buffer
`concentrations so as to maintain the ionic strength at 15 mM
`at respective pHs, without the addition of any salt. The BSA
`
`x
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`"2-,
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`1.15
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`RelativeViscosity
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`1.1:
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`1.21
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`1.19
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`1.17
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`1.15
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`1.13
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`I The relative viscosity (r7,,,,)
`Fig.
`of BSA as a function of solution
`pH. The solid square and triangle
`(primary axis) are the m, calculated
`using Eq.
`l3 from the intercept [r7]
`and siope. 14.9)]? reported by
`Tant'ord and Buzzel (2) at
`IO mM
`ionic strength, The asterisk symbols
`(secondary ends) are the n,“ for
`40 mg/ml BSA solution measured
`in this work.
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`Q Springer
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`Page 6 of 16
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`1 «so mumr
`-l- sums/ml
`use so mglml (this “will
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`i
`/' I i 1.38
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`./ .-
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`RelativeViscosity
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`- 1.28
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`- 1.23
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`1.18
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`'- 1.13
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`

`

`| 975
`High Concentration Bovine Serum Albumin Solutiom
`
`solutions were buffer exchanged with the bulier ofintcrest using,r
`Millipore Amicon Ultra eentrifugation tubes. The concentra-
`tions of the sample were determined using UV spectrophoto-
`metry and an absorptivity 010667 (mg/m1.)"cn‘r'l at 200 nm
`(12) for 0.1% BSA solutions. The solution pH was checked for
`each dialyzed satnple. Required concentmtions were prepared
`by dilution with the respective butler. To account for the
`Donnan effect in our experiments, the initial dialysis btrller
`pH was adjusted appropriately so that
`the filial pH after
`dialysis matched the target p11 and desired ionic strength.
`Additionally, at high concentration the protein itself will
`contribute to the ionic strength of the solution; however, the
`contribution of protein to the total ionic strength of solution is
`hard to quantify owing to a number of ionizable residues and
`their respective pKas, which may very well be (lillercnt from
`the intrinsic pKas due to orientation and eonlhrmational
`placement of these residues in the folded state of the pmtcin.
`For the purpose of this work, the lirial ionic strength of the
`solution will be specified as the contribution from hulfer
`species at a particular pH and added salt, if any.
`Further, an ellbrt was made to reproduce only a part of
`'1"anforrl's data (2)
`to ascertain a similar trend in dilute
`solution viscosity behavior arising due to electroviseous elfccts.
`For the purpose of these measurements, a sinrilar procedure
`as described by 'l‘anford cl (ll. (2) was followed. The BSA was
`dissolved in triple distilled wate ’ and extensively exchanged
`against D1 water using Amicon Ultra eentrifugation tubes.
`Following this, the solution p11 was adjusted using 0.1 N HG]
`or NaOH to the desired pH and final concentration of
`110 mg/ml. The solutions were liltered through 0.22 ttm
`Millipore Millex-W syringe lilters and centrilirged at 6,740
`x g for 5 min rising an eppendorf minispin (Hamburg,
`Germany) centrifuge before making measurements.
`
`Zeta Potential Analysis
`
`Zeta potential (C) measurements were performed at 25t
`0.1°C using a Malvern Zetasizcr Nano Series (Worcester-
`shire, UK) and D'I'SlOGO clear disposable folded capillary
`cell. The methtxlology was kept consistent as detailed in a
`previous work.(l 1) The measured electrophoretie mobility
`was used to determine the 4‘ using Henry’s equation:
`
`
`
`UH:
`
`(l)
`
`where U]; is the electroplroretic mobility under the applied
`voltage, 6 is the dielectric constant of the medium, I] is the
`viscosity of the dispersant, f is the zeta potential in Volts
`andfltjca) is the Henry’s lituction. '1'hcfi(Ka) is a function of
`the electrical double layer around the particle (13,14). At
`15 mM solution ionic strength, the/10m) valtte of 10-15 has
`been used to calculate the f.
`
`Page 7 of 16
`
`Viscosity/Rheological Analysis
`
`For dilrrte solutions (40 mg/ ml 153A) a similar methodology
`as described by 'I'anford cl (:1. was followed (2). The relative
`[low times were measured using a Carrnon-Mar'nring Semi-
`miero Size-25 capillary viscometer (Cannon Instrument
`Company, State College, PA). All the measurements were
`performed using the same viscometer at 251'0.1°C. After
`each measurement the viscometer was cleaned immediately
`with hot sulfuric acid-dichrormrte solution, rinsed numerous
`times to remove all
`traces of the acid, and dried with
`filtered air. Flow times were recorded within 1/100lh of a
`second by means of electric timers. Four to live 11th time
`measurements were made for each solution pH.
`
`the
`For high concentration solutions (250 tug/ml),
`sample viseosities were measured using a VlSCOlab 5000
`viscometer system (Cambridge Viscosity, Medlord, MA). A
`detailed procedure for measurement using VISCOlab 5000
`was described in a previous work (11). The dynamic
`viscosities were determined at 251‘0.1°C by measuring
`the average travel
`time of the pistons calibrated over
`viscoxity ranges 0.5—10.0 el’, 2.5—50 CI’ and 5 -—100 cl’. All
`the samples were analyzed in triplicate. Note that
`the
`V [SC-Olab 5000 is a constant stress viscometer. However,
`the shear rate applied can be calculated by taking into
`account the applied stress, piston and annulus dimensions,
`lhc two way stroke and two way travel time of the piston
`(15,16). For the pistons employed for this study the shear
`ate ranged from 350 to 1,000 Hz. The BSA solutions,
`however, do not show a shear rate dependence up to a
`concentration of 401 mg/ml and 41,700 H7. (17). Before
`each measurement,
`the sample chamber was thoroughly
`cleaned with double-distilled water and dried with nitrogen.
`The rheological properties of BSA were evaluated using
`an ult ‘asonic shear rheometer with quartz crystals vibrating
`at a fundamental frequency of 10 MHZ. The theory and
`experimental procedure have been described previously
`(18). For non-Newtonian viscoelastie fluids,
`the solution
`storage (0') and loss (0") moduli and the complex viscosity
`01*) cart be related to the shift in electrical properties of the
`quartz crystal, i.e. series resistance (R2) and reactance (X2),
`by the following relationships (18):
`2
`,2
`c" (o = #1
`(
`)
`A-p1jr}.
`
`(2)
`
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`QR-X-i
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`{(0) = ([22
`h
`I Pm,
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`./ 2
`,u 2 ”2
`n*= (to) +(6 ))
`
`/w=0*/w
`
`(3)
`
`(4‘)
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`@ Springer
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`

`

`I976
`Yadav. Shire and Kalonia
`
`where A is a crystal constant, pm is the liquid density, and w
`is the quartz crystal frequency. In this study, 35-ttl. samples
`of the BSA solution were analyzed in triplicate.
`
`Dynamic Light Scattering
`
`DLS studies were conducted at 25i0.l°C using a Malvern
`Zetasizer Nano Series (\Norcestcrshire, UK) as described
`previously (1 l). Alter buffer exchange, the protein solutions
`were filtered through 0.22 ttm Millipore Millex-VV syringe
`filters and centrifuged at 6,740 x g for 5 min using an
`epperrdorf minispin (Hamburg, Germany) centrifuge. The
`Zetasizer Nano S utilizes a 632.8 nm Helium-Neon laser
`
`and analyzes scattered light at an angle of 173° using an
`avalanche photodiode. The DTS software was used to
`analyze the acquired correlogram (correlation function
`versus
`time) anti obtain tlte mutual dilfusion cocilicient
`(1),“), which can be expressed as a function of solution
`concentration using the following equation (19):
`
`1),, = D,(l + km)
`
`(5)
`
`is the sclf-dillirsion ctwlficient (the value of D“, at
`where 1),,
`infinite dilution as c—t 0) (20), lrn is the interaction parameter,
`and c is the concentration of the protein (g/ml). The value of
`D, and It!) can be obtained, respectively, from the intercept
`and slope ofa plot of D.“ vs. a (Eq. 5). A positive value of the
`In) corresponds to intermolecular
`rcpulsions, whereas a
`negative k1) signifies attractive interactions between mole-
`cules. The hydrodynamic radius (Rh) ofthe molecules can be
`estimated from the D, using the Stokes-Einstein equation,
`D,=/r.;'l‘/6m]1t’.., where, k3 is the Boltzmann constant, Tis
`the temperature in Kelvin, i] is the solvent viscosity, i.c. c—>0.
`
`Static Light Scattering
`
`