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`Pharmaceutical research.
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`v. 28, nor 8 (Aug‘ 2011)
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`W1 PH167H
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`2011—08—24 10:39:42
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`7"
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`R E S E A R C H
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`journal of the American ASSociation of Pharmaceutical Scientists
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`@_ Springer
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`II095 | ISSN 0724-874!
`28(8) l785-2058 (2m I)
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`Page 1 of 16
`
`CSL EXHIBIT 1067
`
`CSL V. Shire
`
`Page 1 of 16
`
`CSL EXHIBIT 1067
`CSL v. Shire
`
`

`

`Pharmaceutical Research
`
`Volume 28 · Number 8 · August 2011
`
`PERSPECTIVES
`
`Finding Promiscuous Old Drugs for New Uses
`S. Ekins · A.J. Williams 1785
`
`EXPERT REVIEWS
`
`Current and Future Drug Targets in Weight Management
`R.F. Witkamp 1792
`
`Intn•tumoral Drug Delivery with Nanoparticulatc Carriers
`H. 1-Iolback · Y. Yeo 1819
`
`Effects of Surfactants on Lipase Structure, Activity
`and Inhibition
`V. Delorme · R. Dhouib · S. Canaan · F. Fotiadu ·
`F. Carriere· J.-F. Cavalier 1831
`
`Barriers to Non-Viral Vector-Mediated Gene Delivery
`in the Nervous System
`F. C. Perez-Martinez · J. GueJTa · I. Posadas · V. Cena 1843
`
`RESEARCH PAPERS
`
`Validating New Tuberculosis Computational Models
`with Public Whole-Cell Screening Aerobic
`Activity Datasets
`S. Ekins · J.S. Freundlich 1859
`
`Enhanced Oral Bioavailability of Vinpocetine Through
`Mechanochemical Salt Formation: Physico-chemical
`Characterization and In Vivo Studies
`D. 1-Iasa · D. Voinovich · B. Perissutti · M. Grassi ·
`A. Bonifacio · V. Sergo · C. Ccpek · M.R. Chierotti .
`R. Gobctto · S. Dall'Acqua · S. Invernizzi 1870
`
`Aggregation Stability of a Monoclonal Antibody
`During Downstream Processing
`P. Arosio · G. Barolo · T. Miillcr-Spath · H. Wu ·
`M. Morbidelli 1884
`
`Effect of Formulation- and Administration-Related
`Variables on Deposition Pattern of Nasal Spray Pumps
`Evaluated Using a Nasal Cast
`V. Kundoor · R.N. Dalby 1895
`
`Rcgioselcctive Glucuronidation of Flavonols by Six
`Human UGT1A lsoforms
`B. Wu · B. Xu · M. Hu 1905
`
`Laser-Engineered Dissolving Microneedle
`Arrays for Transdcrmal Macromolecular
`Drug Delivery
`K. Migalska · D.I.J. MoiTow · M.J. Garland· R. Thakur·
`A.D. Woolfson · R.F. Donnelly 1919
`
`Investigation of the Pharmacokinetics
`of Romiplostim in Rodents with a Focus
`on the Clearance Mechanism
`Y.-M.C. Wang · B. Sloey · T. Wong · P. Khandelwal ·
`R. Melara · Y.-N. Sun 1931
`
`Using Partial Area for Evaluation of Bioavailability
`and Bioequivalcnce
`M.-L. Chen · B. Davit · R. Lionberger · Z. Wahba ·
`H.-Y. Aim· L.X. Yu 1939
`
`A New Exact Test for the Evaluation of Population
`Pharmacokinetic and/or Pharmacodynamic Models
`Using Random Projections
`C.M. Laffont ·D. Concordet 1948
`
`Clinical Relevance of Liquid Chromatography Tandem
`Mass Spectrometry as an Analytical Method
`in Microdose Clinical Studies
`N. Yamane· Z. Tozuka · M. Kusama · K. Maeda· T. Ikeda·
`Y. Sugiyama 1963
`
`Viscosity Analysis of High Concentration Bovine Serum
`Albumin Aqueous Solutions
`S. Yadav · S.J. Shire · D.S. Kalonia 1973
`
`~Springer
`
`Page 2 of 16
`
`

`

`Poly(Lactide-co-Glycolide) N anocapsules 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 ·
`D.R. de Araujo · E. de Paula · A.H. Rosa ·
`L.F. Fraceto 1984
`
`How Stealthy are PEG-PLA Nanoparticles'? An NIR
`In Vtvo Study Combined with Detailed Size Measurements
`A. Schiidlich ·C. Rose· J. Kuntsche · H. Caysa ·
`T. Mueller· A. Gopferich · K. Mader 1995
`
`Albumin-Coated Porous Hollow Poly(Lactic-co-Glycolic
`Acid) Microparticles Bound with Palmityi-Acylated
`Exendin-4 as a Long-Acting Inhalation Delivery System
`for the Treatment of Diabetes
`H. Kim· J. Lee· T.H. Kim· E.S. Lee· K.T. Oh · D.H. Lee·
`E.-S. Park · Y.H. Bae · K.C. Lee · Y.S. Youn 2008
`
`Formulation, Biological and Pharmacokinetic Studies
`of Sucrose Ester-Stabilized Nanosuspensions
`of Oleanolic Acid
`W. Li · S. Das · K.-y. Ng · P.W.S. Heng 2020
`
`The Role of Constitutive Androstane Receptor
`in Oxazaphosphorine-Mediated Induction
`of Drug-Metabolizing Enzymes in Human Hepatocytes
`D. Wang · L. Li · J. Fuhrman · S. Ferguson ·
`H. Wang 2034
`
`Increased Liver Uptake and Reduced Hepatic Stellate
`Cell Activation with a Cell-Specific Conjugate
`of the Rho-kinase Inhibitor Y27632
`M.M. van Beuge · J. Prakash · M. Lacombe · E. Post ·
`C. Reker-Smit · L. Beljaars · K. Poelstra 2045
`
`AAPS Connection 2055
`
`Cover Image: from the article "Albumin-Coated Porous Hollow
`Poly(Lactic-co-Glycolic Acid) Microparticles Bound with Palmityl(cid:173)
`Acylated Exendin-4 as a Long-Acting Inhalation Delivery System for
`the Treatment of Diabetes," pp. 2008-2019
`
`Further articles can be found at www.springerlink.com.
`
`Abstracted/Indexed in Hygiene and Communicable Diseases.
`Academic Search Alumni Edition, Academic Search Complete,
`Academic Search Premie1;· BIOS!S Previews, Catalysts and
`Catalysed Reactions, CivssFire Beilstein, CSA Biological Sciences,
`CSA Newvsciences Abstracts, Current Abstracts, DaiiJ' Science
`Abstracts, ElvfBASE, Forest Pmducts Abstracts, Global Health.
`Google Scholm; Helmintlzalogical Abstracts, Horticultural Abstracts,
`Horticultural Science Abstracts, !BIDS Index lf!terinarius, IN!S
`Atominde.x, INPHARlvfA, International Pharmaceutical Abstracts,
`Journal Citation Reports/Social Sciences Edition, lvfass S]JectmmetJ:v
`Bulletin, Nutrition Abstracts and Reviews Series A, OCLC ArticleFil:~t
`Database, OCLC FirstSemeh Electmnic Collections Online, B1SCAL,
`Pig News and Information, Postharvest News and li?formation, Potato
`Abstracts, Pmtozoological Abstracts, PubJ\1ed/Med!ine, Review ol
`Agricultural Entomolog;; Review of Ammatic and !vfedicinal Plams.
`Review of lvfedical and Ttterinmy Entomolog;; Review (Jj}v!edical and
`Teterinary Mycology, Science Citation Index, Science Citation Index
`Expanded (SciSeweh), SCOPUS, Sugar lndustJ)' Abstracts, Summon
`by Serial Solutions, TOC Premie1; Teterinwy Bulletin.
`
`Instructions for authors for Pharm Res are available at www.
`springeccom/11095.
`
`Page 3 of 16
`
`

`

`PHARMACEUTICAL RESEARCH
`An Official Journal of the American Association of I•harmaceutical Scientists
`
`Plwrmaceuti~al Research, <~n official journal of the American Association of Pharmaceutical Scientists, publishes papers on innovative research spanning the entire spectrum of
`science that IS the loundation ol. drug diS~overy, development, evaluation, and regulatory approval. Small drug molecules, biotechnology products including genes, pep tides,
`proteins and vaccmes, and gcnellcally engmeered cells arc an mtegral part of papers published in Pharmaceutical Research. Current emphasis of the journal includes, but is not
`limited to, the following areas: !'reformulation; drug delivery and targeting; formulation design, engineering, and processing; pharmacokinetics, pharmacodynamics, and
`pharmacogL!nomics; molecular biopharmaccutics and drug disposition; and computational biopharmaccutics.
`
`EDITOR-IN-CIJIEF
`Peter W. Swaan, University of Maryland, Baltimore, Maryland, USA
`
`EDITORS
`Paul M. Bummer, University of Kentucky, Lexington, Kentucky, USA
`Margareta llanunarlund-Udenaes, Uppsala University, Uppsala, Sweden
`Ken-ichi Jnui, Kyoto Pharmaceutical University, Kyoto, Japan
`Wim Jiskoot, Leidcn University, Netherlands
`Uday Kompella, University of Colorado, Denver, Colorado, USA
`Ah-Ng Tony Kong, State Univasity of New Jersey-Rutgers, Piscataway,
`New Jersey, USA
`Tamara Minko, State University of New Jersey-Rutgers, Piscataway, New Jersey, USA
`.James E. Polli, University of Maryland, llaltimore, Maryland, USA
`David E. Smith, University of Michigan, Ann Arbor, Michigan, USA
`Christine Vauthicr, Centre National de Ia Recherche Scientifique, Paris, France
`Ernst Wagner, Ludwig-Maximilians-Universitiit, Munich, Germany
`Yuhong Xn, Shanghai Jiao Tong University Mcd-X Research Institute,
`Shanghai, China
`
`EDITOR, EXPERT REVIEWS
`Scan Ekins, Collaborations in Chemistry, Philadelphia, Pennsylvania, USA
`
`EDITOR, SI'ECIAL I•'EATURES
`Ram 1. Mahato, University of Tennessee, Memphis, Tennessee, USA
`
`CONSULTING EDITORS
`Per Artursson, Uppsala University, Uppsala, Sweden
`.Jessie L-S. Au, Ohio State University, Columbus, Ohio, USA
`Daan J.A. Crommelin, Utrecht Institute for Pharmaceutical Sciences,
`Utrecht, Netherlands
`Wim E. Hennink, Utrecht Institute for Pharmaceutical Sciences,
`Utrecht, Netherlands
`William Jusko, SUNY lluffalo, lluffalo, New York, USA
`Nicholas A. J'CPI•as, University of Texas at Austin, Austin, Texas, USA
`Joseph w. Polli, Gla~oSmithKlii~e, R~search Triangle Park, North Carolina, USA
`Wolfgang Sadce, Ohio State Umversity, Columbus, Ohio, USA
`Danny Shen, University of Was~Iington •. ~cattle, Washington, USA
`Yuichi Sugiyama, Umversity of fokyo, Iokyo, Japan
`Bernard Testa, University Hospital Center, Laussane, Switzerland
`
`EDITORIAL ADVISORY HOARD
`Sandra R.n. Allerhciligen, Eli Lilly, Indianapolis, Indiana, USA
`Bradley J). Anderson, Uni~ersity ~f Kcnt~cky, Lexington, Kentucky, USA
`Patrick Augustijns, Kathohcke UmversitCit Leuven, Leuven, llelgium
`You-Han Hac, University of Utah, Salt Lake City, Utah, USA
`Reina Jlcndayan, University of Toronto, Toronto, Canada
`Andreas Jlernko11·Schniirch, University of Innsbruck, Innsbruck, Austria
`Robin 11. Bogner, University of Connecticut, Storrs, Connecticut, USA
`Kathleen M.K. Uojc, SUNY l3uffalo, lluffalo, New York, USA
`Youugro Byun, Seoul ~ati<~nal University, Seoul, Korea
`.John [<'. Carpenter, UmverSity of Colorado, Denver, Colorado, USA
`Jlak Kim Chan, University of Sydney, Sydney, Australia
`Albert JI,L. Chow, Chinese University of Hong Kong, Sha Tin, Hong Kong
`Paolo Colombo, University of Parma, Parma, Italy
`.James T. Dalton, Ohio State University, Columbus, Ohio, USA
`Gerard Dclcris, University of llordeaux, llordcaux, France
`William F. EJn)(JUist, University of Minnesota, Minneapolis, Minnesota, USA
`Gcrt Fricker, Ruprecht-Karls-Univcrsitat Heidelberg, Heidelberg, Germany
`Lawrence Gan, MillcnniumPharmaccuticals, Inc., Cambridge, Massachusetts, USA
`
`Vadivcl Gmmpathy, Medical College of Georgia, Augusta, Georgia, USA
`Bruno Gander, ETI-I Ziirich Institute of Pharmaceutical Sciences,
`Ziirich, Switzerland
`llmnidreza Ghandchari, University of Utah, Salt Lake City, Utah, USA
`Scott Grossman, llristol-Myers Squibb, Wallingford, Connecticut, USA
`Hyo-Kyung Han, Dongguk University, Seoul, Korea
`Bruno C. lhmcock, Pfizer Inc., Groton, Connecticut, USA
`llideyoshi Harashima, 1-Iokkaido University, Sapporo, Japan
`Jan Haworth, University of Southern California, Los Angeles, California, USA
`Anthony J. Jlickey, University of North Carolina at Chapel Hill, Chapel Hill,
`North Carolina, USA
`Kathleen Ilillgren, Eli Lilly, Indianapolis, Indiana, USA
`Gunther llochhaus, University of Florida, Gainesville, Florida, USA
`Nicholas H.G. Holford, University of Auckland, Auckland, New Zealand
`Ken-ichi Hosoya, Toyama University, Toya.ma, !apa~ .
`. , .
`Jin-Ding Huang, National Cheng-Kung UmverSity, 1 aman, I mwan
`Aj<lz Hussain, Philip Morris International, Washington, D.C., USA
`Toshihiko Ikeda, Sankyo Co. Ltd., Tokyo, Japan
`Alcx<mder V. Kabanov, University of Nebraska, Omaha, Nebraska, USA
`lliroyuki Kusulmra, University of Tokyo, To~yo, Japan
`.
`Glen S. Kwon, University of Wisconsin, Madison, Wisconsm, USA
`Claus-Michael Lehr, Saarland University, Saarbrueken, Germany
`Tonglei Li, University of Kentucky, Lexington, Kentucky, USA
`.
`Jiunn H. Lin, Merck Research Laboratories, Westpmnt, P~nnsylvama, USA
`Dexi Lin, University of Pittsburgh, Pittsburgh, Pennsylvama, USA
`Zheng-Rong Lu, University of Utah, Salt Lake City, Utah, USA
`Panos Macheras, University of Athens, Athens, Greece
`l'atrick McNamara, University of Kentucky, Lexington, Kentucky, USA
`David Mooney, Harvard University, Cambridge, Massachusetts, USA
`Marilyn E. Morris, SUNY Buffalo, Buffalo, New York, USA
`Emi Nakashima, Kyroitsu University, Tokyo, Japan
`Tac Gwan l'ark, Korea Advanced Institute of Science and Technology,
`Deajon, Korea
`.
`Chris Porter, Monash University, Melbourne, Australia
`Mark Prausnitz, Georgia Institute of Technology, Atlanta, Georgia, USA
`David Putnam, Cornell University, Ithaca, New. York, USA
`.
`Michael Roberts, University of Queensland, llnsbane, Australia
`l'atrizia Santi, University of Parma, Panna, Italy
`Chang-K110 Shim, Seoul National Universit~, Seoul,Kon:a
`Steven J. Shire, Genentech, Inc., San FranciSco, Cahforma, USA
`Audra L. Stinchcomb, University of Kentucky, Lexmgton, Kentucky, USA
`Hiroshi Suwki, University of Tokyo Hospital, Tokyo, Japan
`Yoshinobu T:1kakura, Kyoto University, Kyoto, Japan
`Ikumi Tamai, Kanazawa University, Kakuma, Japan
`.
`Jiiaki K Troconiz, University of Navarra, Pamplona, Spam
`Kishor M. Wasan, University of llritish Columbia, Vancouver, Canada
`Ronald E. White, Schering-Plough Research Institute, Kenilworth,
`New Jersey, USA
`.
`Guillaume Wientjes, Ohio State University, Columbus, Ol~IO, USA
`Wen Xie, University of Pittsburgh, Pittsburgh, Pennsylvama, USA
`Keiji Yamamoto, Chiba University, Chiba, Japan
`.
`Fmniyoshi Yamashita, Kyoto University, Kyoto, Japan
`Guofcng You, State University of New Jersey- Rutgers, Piscataway,
`New Jersey, USA
`
`EI>ITORIAL ASSISTANT
`Rachel n. Lucke, University of Maryland, llaltimore, Maryland, USA
`
`Page 4 of 16
`
`

`

`Pharm Res (20 I I) 28: 1973-1983
`DOl I 0.1 007/s II 095-011-0424-7
`
`RESEARCH PAPER
`
`Viscosity Analysis of High Concentration Bovine Serum
`Albumin Aqueous Solutions
`
`Sandeep Yadav ·Steven J. Shire· Devendra S. Kalonia
`
`Received: S December 201 0 I Accepted: 8 March 2011 I Published online: 14 April 2011
`((] Springer Science+ Business Media, LLC 2011
`
`ABSTRACT
`Purpose To understand the apparent inconsistency between
`the dilute and high concentration viscosity behavior of bovine
`serum albumin (BSA).
`Method Zeta potential and molecular charge on BSA were
`determined from Electrophoretic mobility measurements.
`Second virial coefficient (8n) and interaction parameter (k0 )
`obtained from static and dynamic light scattering, respectively,
`quantified intermolecular interactions. Rheology studies char(cid:173)
`acterized viscoelasticity at high concentration. The dipole
`moment was calculated using Takashima's approximation for
`proton fluctuations over charged residues.
`Results The effective isoelectric point of BSA was pH 4.95. In
`dilute solutions (:S 40 mg/ml), the viscosity was minimal at the
`pi; at high concentrations, pH 5.0 solutions were most viscous.
`8 22 and k0 showed intermolecular attractions at pH 5.0;
`repulsions dominated at other pHs. The attractive interactions
`led to a high storage modulus (G') at pH 5.0.
`
`Electronic supplementary material The online version of this article
`(doi: I 0.1 007/s I I 095-0 I 1-0424-7) contains supplementary material,
`which is available to authorized users.
`
`S. Yadav · D. S. Kalonia (!8J)
`Department of Pharmaceutical Sciences, University of Connecticut
`Storrs, CT 06269, USA
`e-mail: kalonia@uconn.edu
`
`S. J. Shire
`Late Stage Pharmaceutical Development
`Genentech, Inc.
`I DNAWay
`South San Francisco, CA 94080, USA
`
`Present Address:
`S. Yadav
`Late Stage Pharmaceutical Development
`Genentech, Inc.
`South San Francisco, CA 94080, USA
`
`Conclusion In dilute solutions, the electroviscous effect due to
`net charge governs the viscosity behavior; at high concen(cid:173)
`trations, the solution viscosity cannot be justified based on a
`single parameter. The net interplay of all intermolecular forces
`dictates viscosity behavior, wherein intermolecular attraction
`leads to a higher solution viscosity.
`
`KEY WORDS dipole moment· high concentration viscosity·
`interaction parameter (k0 ). · intermolecular interaction · protein
`charge · second vi rial coefficient (8 22) ·zeta potential
`
`INTRODUCTION
`
`In 1956 Buzzcl and Tanford published the viscosity of
`bovine serum albumin (BSA) and ribonuclease (RNAasc) at
`various conditions of solution pH and ionic strengths (I ,2).
`For the concentration range studied (-40-50 mg/ml) the
`solution viscosity showed a good correlation with the net
`charge-induced clectroviscous eflects. Due to the presence
`of electrical charge 011
`the molecule, three kinds or
`contribution may affect the viscosity behavior. A 'prirmuy
`effect' due to the resistance of the diffuse double layer
`surrounding the molecule, a 'secondary eflcct' due to the
`intermolecular repulsion between double layers and a
`'tertiary ellcct' that may arise if the interparticle repulsion
`affects the shape of the macromolecule. These three arc
`collectively known as the 'electroviscous cllects' (3). vVhen a
`charged particle moves through a medium comprising
`small ions, electrostatic interaction between the particle
`and the small ions results in a relative motion of the ions to
`the medium and consequently an additional viscous loss
`arises that contributes to the overall viscosity of the solution.
`For BSA and RNAasc solutions the slope of the reduced
`viscosity (1/rcd), i.e. the specific increment in viscosity as a
`
`~Springer
`
`Page 5 of 16
`
`

`

`1974
`
`Yadav, Shire and Kalonia
`
`limction of protein concentration (c), increased with an
`increase in molecular charge ( 1 ,2). An increase in solution
`ionic strength resulted in a decrease in the slope (17,cd versus 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 elcctroviscous cllccts,
`which also correlated to some extent with Booth's theory (4)
`[17] clue to
`of anticipated increase in intrinsic viscosity
`electroviscous ellccts ( 1 ,2).
`The observed behavior suggests that the viscosity should
`increase with an increase in the molecular charge due to the
`additional resistance to !low ollcrcd by the electroviscous
`effects. It then follows that for a protein solution the
`viscosity should be minimal at the isoelcctric point (pi),
`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). Furthermore, for BSA solution,
`certain calculations have been presented using the orit,rinal data
`in Tanford's work (2), which supports this argument (Fig. 1).
`The details of the calculation for Fig. I 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
`clillcrent behavior. The viscosity for 120 mg/ml IgG2
`solution was observed to be highest at the pi (7), which is
`not in agreement with the net charge-induced electro(cid:173)
`viscous effects. Conversely, 130 mg/ml MAb-1 (IgG 1), with
`a measured pi of7.8, showed the highest viscosity at pH 6.0
`relative to other pHs studied (8,9). Salinas et al. suggested
`that the high viscosity observed for >50 mg/ml IgG 1
`solution, at pH 6.0 was primarily due to electroviscous
`effects (I 0). On the contrary, Yadav et al. (II) did not
`observe a consistent interpretation of electroviscous ellccts
`to the viscosity behavior or four dillerent MAbs. (11)
`The dilute solution viscosity behavior of BSA, wherein
`the viscosity was observed to be minimal at the pi (Fig. I),
`
`therefore, does not correlate with the recently published
`high concentration viscosity data on IgG molecules. The
`present study seeks to understand the apparent inconsistency
`between the dilute am! high concentration viscosity behavior
`of protein solutions. In particular, the high concentration
`viscosity behavior of BSA solutions as well as the dificrent
`l~tctors that may be responsible for the observed behavior
`have been analyzed and discussed.
`
`MATERIALS AND METHODS
`
`Materials
`
`The BSA (lyophilized, purity 99% and essentially fatty acid(cid:173)
`and globulin-free (Catalogue number: A0281) was obtained
`from Sigma (St. Louis, MO). All other chemicals including
`acetic acid, sodium acetate, sodium chloride, histidine
`hydrochloride, monobasic and dibasic sodium phosphate,
`were obtained from Fisher Scientific (Fair Lawn, Nj). All
`chemicals used were reagent grade or higher. Deionized
`water equivalent to Milli-Qri\l grade was used to prepare
`all solutions. Millipore (Billerica, MA) Amicon Ultra
`centrifugation tubes with a molecular weight cut-off of
`3 kD were obtained from Fisher Scientific. Quartz crystal
`discs with fundamental vibrating frequencies of I 0 MHz
`and plated with gold electrodes on both sides were
`obtained from International Crystal Manuf~tcturing
`Company (Oklahoma City, Oklahoma).
`
`Methods
`
`Acetic acid-sodium acetate (pH 4.0, 5.0), histidine hydrochlo(cid:173)
`ride (pH 6.0) and monobasic-dibasic sodium phosphate (pH 7.0
`and 8.0) hullers wer~ prepared with appropriate buffer
`concentrations so as to maintain the ionic strength at 15 ml\1
`at respective pHs, without the addition of any salt. The BSA
`
`Fig. I The relative viscosity (fJ,e~)
`of BSA as a function of solution
`pH. The solid square and triangle
`(primary axis) are the f),u calculated
`using Eq. I 3 from the intercept [f)]
`and slope, kH[ fJf, reported by
`Tanford and Buzzel (2) at I 0 mM
`ionic strength. The asterisk symbols
`(secondary axis) are the fJrel for
`40 mg/ml BSA solution measured
`in this work.
`
`1::) Springer
`
`1.25
`
`1.23
`
`1.21
`
`1.19.
`
`1.17
`
`1.15
`
`~ ·;;;
`~
`>
`"' ·~ Ill
`
`Qj
`a:
`
`)!\.._
`
`-II ·40 mg/ml
`
`-&-30mg/ml
`··Jr.·· 40 mg/ml (this study)
`
`.. ········
`·~··························)K"
`
`... ·1.38
`
`.. X~ 1.33
`.... ··
`
`.... ···
`
`1.28
`
`~ ·;;;
`~
`>
`"' ·~ Ill
`
`.__ ----.. --.,.----------------------________ .,.
`
`1.23 Qj
`a:
`
`1.18
`
`1.13 +-----.,.--------,,.-------,.-----~ 1.13
`3.5
`4.5
`5.5
`6.5
`7.5
`oH
`
`Page 6 of 16
`
`

`

`High Concentration Bovine Serum Albumin Solutions
`
`1975
`
`solutions were buller exchanged with the buller of interest using
`Millipore Amicon Ultra ccntrifttgation tubes. The concentra(cid:173)
`tions of the sample were determined using UV spectrophoto(cid:173)
`metry and an absorptivity ofO.GG7 (mg/mL)- 1cm- 1 at 2BO nm
`( 12) f(Jr 0.1 °1<> liSA solutions. The solution pH was checked for
`each dialyLed sample. Required concentrations were prepared
`by dilution with the respective buffer. To account for the
`Donnan ellcct in our expetimcnts, the initial dialysis buller
`pH was acljustcd appropriately so that the final pH allcr
`dialysis matched the target pH and desired ionic strength.
`Additionally, at high conccntratiott 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 vety well he dillercnt from
`the intrinsic pKas due to mientation and conf(xmational
`placement of these residues in the folded state of the protein.
`For the purpose of this work, the final ionic strength of the
`solution will be specified as the conttibution from buller
`species at a particular pH and added salt, if any.
`Further, an c!Iort was made lo reproduce only a part of
`Tanford's data (2) to ascertain a sirnilar trend in dilute
`solution viscosity behavior arising due to clcctroviscous ellects.
`For the purpose of these measurements, a similar procedure
`as described by Tanlord et a!. (2) was followed. The BSA was
`dissolved in triple distilled water and extensively exchanged
`against DI water using Amicon Ultra centrifugation tubes.
`Following this, the solution pH was acljusted using 0.1 N HCI
`or NaOH to the desired pH and final concentration of
`40 mg/ml. The solutions were liltcrcd through 0.22 ~tm
`Milliporc Millcx-W syringe liltcrs and centrifuged at 6,740
`x g for 5 min using an cppendorf minispin (Hamburg,
`Germany) ccnttifttge before making measurements.
`
`Zeta Potential Analysis
`
`Zeta potential (!;) measurements were pcrf(Jrmccl at 25 ±
`0.1 oc using a l'vfalvcm Zctasizer Nano Series (vVorcestcr(cid:173)
`shirc, UK) and DTS I 060 clear disposable folded capillaty
`cell. The methodology was kept consistent as detailed in a
`previous work.(! I) The measured electrophoretic mobility
`was used to determine the ?; using Henry's equation:
`
`Viscosity/Rheological Analysis
`
`For dilute solutions (4·0 mg/ml BSA) a similar methodology
`as described by Tanlon.l et al. was followed (2). The relative
`flow times were measured using a Cannon-Manning Semi(cid:173)
`micro Size-25 capillaty viscometer (Cannon Instrument
`Company, State College, PA). All the measurements were
`performed using the same viscometer at 25 ± 0.1 °C. Aller
`each measurement the viscometer was cleaned immediately
`with hot sulfuric acid-dichromate solution, rinsed numerous
`times to remove all traces of the acid, and dried with
`filtered air. Flow times were recorded within l I l OO'" of a
`second by means of electric timers. Four to five flow time
`measurements were made for each solution pH.
`For high concentration solutions (250 mg/ml), the
`sample viscosities were measured using a VTSCO!ab 5000
`viscometer system (Cambridge Viscosity, Medford, MA). A
`detailed procedure for measurement using VISCO lab 5000
`was described in a previous work (11). The dynamic
`viscosities were determined at 25 ± 0.1 oc by measuring
`the average travel time of the pistons calibrated over
`viscosity ranges 0.5-10.0 cP, 2.5-50 cP and 5-100 cP. All
`the samples were analyzed in triplicate. Note that the
`VISCO!ab 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,
`the two way stroke and two way travel time of the piston
`(15,16). For the pistons employed for this study the shear
`rate ranged from 350 to I ,000 Hz. The BSA solutions,
`however, do not show a shear rate dependence up to a
`concentration of 4-04 rng/ml and 4·, 700 Hz (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 ultrasonic shear rheometer with quartz crystals vibrating
`at a ftmdamental frequency of I 0 MHz. The theory and
`experimental procedure have been described previously
`(!B). For non-Newtonian viscoelastic lluids, the solution
`storage (G') and loss (G") moduli and the complex viscosity
`('1*) can be related to the shift in electrical properties of the
`quartz crystal, i.e. series resistance (R2) and reactance (X2),
`by the following relationships (I B):
`
`(!)
`
`where Ug is the electrophoretic mobility under the applied
`voltage, c is the dielectric constant of the medium, 17 is the
`viscosity of the dispersant, ?; is the zeta potential in Volts
`aml}i(Ka) is the Henry's ftmction. Thc}i(lca) is a function or
`the electrical double layer around the particle ( 13, 14-). At
`15 mM solution ionic strength, t!tc.Ji(Ka) value of 1.045 has
`been used to calculate the ?;.
`
`G"(w) = 2~2X2
`A-pLiq
`
`IJ* = ( c't + ( c"t 1 w = G* 1 cv
`
`(
`
`')
`
`')) 1/2
`
`(2)
`
`(3)
`
`(4·)
`
`~Springer
`
`Page 7 of 16
`
`

`

`1976
`
`Yadav, Shire and Kalonia
`
`where A is a crystal constant, Pti<J is the liquid density, and w
`is the quartz crystal frequency. In this study, 35-).lL samples
`of the BSA solution were analyzed in triplicate.
`
`Dynamic Light Scattering
`
`DLS studies were conducted at 25 ± 0. I °C using a Malvern
`Zctasizer Nano Series (Worccstcrshirc, UK) as described
`previously (I I). Aller buflcr exchange, the protein solutions
`were filtered through 0.22 ~Lm Milliporc Millcx-W syringe
`filters and centrifuged at 6, 7 L1Q x g for 5 min using an
`eppenclorf minispin (Hamburg, Germany) ccntril\1ge. The
`Zctasizcr Nano S utilizes a 632.B nm Helium-Neon laser
`and analyzes scattered light at an angle of I 73° using an
`avalanche photodiodc. The DTS software was used to
`analyze the acquired corrclogram (correlation function
`versus time) and obtain the mutual diflusion cocflicicnt
`(Dm), which can be expressed as a function of solution
`concentration using the following equation (I 9):
`Dm = D,( 1 + knc)
`where D, is the sclf-diflitsion coefficient (the value of Dm at
`infinite dilution as c-O) (20), kv is the interaction parameter,
`and cis the concentration of the protein (g/ml). The value of
`D, and ku can be obtained, respectively, from the intercept
`and slope of a plot of Dm vs. c (Eq. 5). A positive value of the
`kn corresponds to intermolecular repulsions, whereas a
`negative kD signifies attractive interactions between mole(cid:173)
`cules. The hydrodynamic radius (Rh) of the molecules can be
`estimated lrorn the D, using the Stokes-Einstein equation,
`D,=knT16nr!Rh, where, ku is the Boltzmann constant, Tis
`the temperature in Kelvin, 17 is the solvent viscosity, i.e. c-O.
`
`(5)
`
`Static Light Scattering
`
`SLS studies were conducted at 25 ± 0.1 °C using a Malvern
`Instruments (vVorccstershire, UK) Zctasizcr Nano S. Sam(cid:173)
`ple preparation steps were similar to that used for DLS.
`Samples were analyzed at I 2 mg/ml and then sequentially
`diluted to lower concentrations. The average scattered
`intensity was obtained using the attenuation-corrected
`derived count rates from the Malvern Zetasizcr (2 I). The
`Debye plots were then constructed from the average
`scattered intensities using the following equation:
`
`KC
`I
`..,- = - - + 2B"JC where the optical constant
`--
`iV!w
`1'?.e
`
`(G)
`
`index increment brought about by the solute under a given
`set of solution conditions.
`Note that the Malvcm Zetasizcr Nano Series (\l\1orcestcrshire,
`UK) uses an Avalanche Plwtodiode detector (APD) for
`recording the scattering intensity signal. Using the APD,
`both the SLS and DLS measurements can be per(cid:173)
`formed simultaneously where the instrument measures
`the time-averaged scattered intensity lor SLS and the
`time-dependent fluctuation
`in scattered
`intensity lor
`DLS, by means of photon counting and photon
`correlation, respectively. However, the instrument uses
`an attenuator for recording the time-dependent fluctu(cid:173)
`ation in scattered intensity while performing the DLS
`measurements, whereas there is no attenuation of the
`excess scattered intensity signal that reaches the detector
`while performing SLS measurements. This results in
`APD saturation
`in SLS measurements resulting
`in
`erroneous results. The correct average scattered inten(cid:173)
`sity can, however, be determined from the attenuation(cid:173)
`corrected count rates from the DLS measurements. A
`to obtain
`detailed procedure for such a correction
`correct SLS parameters using a Malvern Zctasizer is
`discussed elsewhere (2 I).
`
`RESULTS
`
`The asterisk symbol in Fig. I (secondary y-axis) shows the
`relative viscosity of a 4·0 mg/rnl BSA solution rneasurecl
`using a capillary viscometer following a similar procedure
`as described by Tanlord et al. (2) in his original work. At
`40 rng/rnl BSA at pH 5.0 showed a minir'1al viscosity
`compared to other pHs. The solid square and the triangles
`are 40 and 30 mg/ml, respectively, for BSA solution
`viscosities calculated frorn Tanford's work (2) and do not
`represent measurements made in this work. Further details
`on these calculations arc elaborated in the Discussion
`section. Although Tanforcl's data already suggested a
`minimal viscosity around pH 5.0, the measurements vvcrc
`repeated to ensure that the trend observed in viscosity
`behavior, due to clcctroviscous cflccts in dilute solution,
`holds in general and was not an artil~tct of a difrercnt grade
`of BSA used previously in the study of Tanford ct a!. (2) The
`relative magnitudes of viscosity observed in the two studies
`arc diflcrcnt and may be due to dillcrenees in purity of BSA
`obtained from dillerent sources; however, the change in
`viscosity as a function of pH is consistent in the two studies.
`
`(7)
`
`Zeta Potential Measurements
`
`Nfw is the weight average molecular weight of the solute, cis
`the concentration in g/rnl, A0
`is the wavelength of light
`used, Ni\ is Avogadro's number and dn/dc is the rcfi·activc
`
`Figure 2 shows the C: of BSA molecules as a limction of
`solution pH. The point of zero charge or the crossover
`point from a positive to negative potential, referred to as
`
`~Springer
`
`Page 8 of 16
`
`

`

`High Concentration Bovine Serum Albumin Solutions
`
`1977
`
`5.0, the BSA molecule carries a net positive or a negative
`charge, respectively, whereas at the pi (-pH 4.95), the net
`molecular charge is zero. The calculatccl net charge at
`difl'ercnt pH was in good agreement with the reported
`values of mean charge obtained from titration curves (23)
`(Su