Volume 289, Nbs. 1-2,
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`31 JANUARY 2005
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`ISSN 0378-5173
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`·
`Is ernati
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`CSL EXHIBIT 1066
`CSL v. Shire
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`Page 1 of 35
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`INTERNATIONAL JOURNAL OF PHARMACEUTICS
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`AIMS AND SCOPE
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`Jmunic·Itions ami notes dealing
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`he lntemational Journal of Pharmaceutics publishes innovative papers, revtews, n
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`di'lnnostic enllly, me u mg
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`!ton and evaluation of drug delivery systems in vitro and 111 VIVO. Drug
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`oligonucleotides, gene constructs and radiophannaceuticals.
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`reas o! particular mterest mclude: phannaceuttcal nanotechnology; P 1ystc.1 P arni.J
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`.. membrane !unction and
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`, , , t' , ... 1bsorption mechantsms,
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`app ted to pharmaceutics; exctptent functton and charactensatton; btop mnnaceu tcs, • ·
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`lb· k . nd contro mec 1ant.
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`transport; novel routes and modes of delivery; responstvc delivery systems, eet dC
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`(C'Irricr-h<>and mtct.IC t(
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`applicatiOns of cell and molecular biology to drug delivery; prodrug destgn; btoa( lest on
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`(protein and peptide formulation and delivery).
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`EDITORS-IN-CHIEF
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`Editor-in-Chief: A.T. FLORENCE
`Editor: G. BUCKTON
`The School of Pharmacy,
`University or London
`29/39 Brunswick Square
`London WCIN lAX, U.K.
`E-mail: ijp@ulsop.ac.uk
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`J.l!. RYTTING
`Pharmaceutical Chemistry Dept.,
`University or Kansas
`2095 Constant Avenue
`Lawrence, KS 66047
`U.S.A.
`E-mail: ijp@ku.edu
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`Editor-in-Chief: T. NAGAI
`Associate Editor: T. SONOBE
`Dept. of Pharmaceutical Engineering
`School of Pharmaceutical Sciences
`University of Shizuoka 52-I Yada
`Shizuoka-shi 422-8526 Japan
`E-mai 1: sonobe@ u-shizuoka-ken.ac.jp
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`All manuscripts should be addressed to the
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`G. Alderborn (Uppsala, Sweden)
`M.J. Alonso (Santiago de Compostela, Spain)
`D. Attwood (Manchester, U.K.)
`K.L. Audus (Lawrence, KS, U.S.A.)
`G. Borchard (Piscataway, NJ, U.S.A.)
`J. Bouwstra (Leiden, The Netherlands)
`H.-K. Chan (Sydney, NSW, Australia)
`W.N. Channan (!'arkville, Australia)
`J.H. Collett (Manchester, U.K.)
`D.Q.M. Craig (Norwich, U.K.)
`D..T.A. Cromrnelin (Utrecht, The Netherlands)
`D. Duchene (Chatenay-Malabry, France)
`J.L. Ford (Liverpool, U.K.)
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`EDITORIAL BOARD
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`A. Lloyd (Brighton, U.K.)
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`R.H. MUller (Berlin, Germany)
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`Volume 289/1 2 (2005)
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`INTERNATIONAL
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`PHARMACEUTICS
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`Editors-in-Chief
`A.T. FLORENCE (London, U.K.)
`T. NAGAI (Tokyo, Japan)
`J.H. RYTTING (Lawrence, KS, U.S.A.)
`
`Editor
`G. BUCKTON (London, U.K.)
`
`Associate Editor
`T. SONOBE (Shizuoka-shi, Japan)
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`Editorial Board
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`G. ALDERBORN (Uppsala, Sweden)
`M.J. ALONSO (Santiago de Compostela, Spain)
`D. ATTWOOD (Manchester, U.K.)
`K.L. AUDUS (Lawrence, KS, U.S.A.)
`G. BORCHARD (Piscataway, NJ, U.S.A.)
`J. BOUWSTRA (Leiden, The Netherlands)
`H.-K. CHAN (Sydney, NSW, Australia)
`W.N. CHARMAN (Parkville, Australia)
`J.H. COLLETT (Manchester, U.K.)
`D.Q.M. CRAIG (Norwich, U.K.)
`D.J.A. CROMMELIN (Utrecht, The Netherlands)
`D. DUCHENE (Chfitenay-Malabry, France)
`J.L. FORD (Liverpool, U.K.)
`J. HADGRAFT (London, U.K.)
`H. HARASHlMA (Hokkaido, Japan)
`W.l. HIGUCHI (Salt Lake City, UT, U.S.A.)
`L. JLLUM (Nottingham, U.K.)
`H.G. KRISTENSEN (Copenhagen, Denmark)
`A. LLOYD (Brighton, U.K.)
`T. LOFTSSON (Reykjavik, Iceland)
`P. MACHERAS (Athens, Greece)
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`Y. MAITANI (Tokyo, Japan)
`G.P. MARTIN (London, U.K.)
`A.K. MITRA (Kansas City, MO, U.S.A.)
`R.H. MULLER (Berlin, Germany)
`J.M. NEWTON (London, U.K.)
`N. PEPPAS (Austin, TX, U.S.A.)
`F. PODCZECK (Sunderland, U.K.)
`J.P. REMON (Gent, Belgium)
`A.J. REPTA (San Carlos, CA, U.S.A.)
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`E. SHEFTER (San Diego, CA, U.S.A.)
`K. SUGIBAYASHI (Saitama, Japan)
`Y. SUGIYAMA (Tokyo, Japan)
`!.G. TUCKER (Dunedin, New Zealand)
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`K. UEKAMA (Kumamoto, Japan)
`A. URTTI (Kuopio, Finland)
`S.P. VYAS (Sagar, India)
`D.E. WURSTER (Iowa City, lA, U.S.A.)
`S. YALKOWSKY (Tucson, AZ, U.S.A.)
`A. YAMAMOTO (Kyoto, Japan)
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`ELSEVIER
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`Amsterdam • Boston • Jena • London • New York • Oxford • Paris • Philadelphia • San Diego • St. Louis
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`VOL. 289/1-2 (2005)
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`doi: 10.10 16/S0378-5173(04)00750-l
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`Page 5 of 35
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`International Journal of Pharmaceutics 289 (2005) 1–30
`
`Review
`Protein aggregation and its inhibition in biopharmaceutics
`∗
`
`Wei Wang
`
`Biotechnology Division, Bayer HealthCare, 800 Dwight Way, Berkeley, CA 94701, USA
`
`Received 13 May 2004; received in revised form 20 August 2004; accepted 12 November 2004
`
`Abstract
`
`Protein aggregation is arguably the most common and troubling manifestation of protein instability, encountered in almost
`all stages of protein drug development. Protein aggregation, along with other physical and/or chemical instabilities of proteins,
`remains to be one of the major road barriers hindering rapid commercialization of potential protein drug candidates. Although
`a variety of methods have been used/designed to prevent/inhibit protein aggregation, the end results are often unsatisfactory
`for many proteins. The limited success is partly due to our lack of a clear understanding of the protein aggregation process.
`This article intends to discuss protein aggregation and its related mechanisms, methods characterizing protein aggregation,
`factors affecting protein aggregation, and possible venues in aggregation prevention/inhibition in various stages of protein drug
`development.
`© 2004 Elsevier B.V. All rights reserved.
`
`Keywords Protein aggregation; Aggregation mechanism; Protein refolding; Protein formulation; Protein stabilization
`
`Abbreviations A␤, amyloid ␤ peptide; BSA, bovine serum albumin; CAB, carbonic anhydrase B; CD, circular dichroism; rConIFN,
`recombinant consensus ␣-interferon; CspA, cold shock protein A; rhDNase, recombinant human deoxyribonuclease; DSC, differential scan-
`ning calorimetry; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast
`growth factor; rFVIII, recombinant factor VIII; rFIX, recombinant factor IX; rFXIII, recombinant factor XIII; rhGCSF, recombinant human
`granulocyte colony stimulating factor; GDH, glutamate dehydrogenase; pGH, porcine growth hormone; rhGH, recombinant human growth
`hormone; GdnHCl, quanidine hydrochloride; GSH, reduced glutathione; GSSG, oxidized glutathione; rHA, recombinant human albumin; HP-
`␤-CD, hydroxypropyl-␤-cyclodextrin; HSA, human serum albumin; IFN-␤, interferon-␤; IFN-␥, interferon-␥; IgG, immunoglobulin G; IL-1␤,
`interleukin-1␤; IL-2, interleukin-2; rhIL-1ra, recombinant human interleukin-1 receptor antagonist; IR, infrared spectroscopy; rhKGF, recombi-
`nant human keratinocyte growth factor; LDH, lactate dehydrogenase; LMW-UK, low molecular weight urokinase; Mab, monoclonal antibody;
`rhMGDF, recombinant human megakaryocyte growth and development factor; NMR, nuclear magnetic resonance spectroscopy; PAGE, poly-
`acrylamide gel electrophoresis; PBS, phosphate buffered saline; PEG, polyethylene glycol; PVA, polyvinyl alcohol; RH, relative humidity;
`RP-HPLC, reversed phase HPLC; RNase A, ribonuclease A; TNF, tumor necrosis factor; tPA, tissue plasminogen activator; SDS, sodium
`dodecyl sulfate; SEC-HPLC, size exclusion HPLC
`∗
`Present address: Pfizer Inc., 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA. Tel.: +1 636 247 2111; fax: +1 636 247 5030.
`E-mail address wei.2.wang@pfizer.com.
`
`0378-5173/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
`doi:10 1016/j.ijpharm.2004.11.014
`
`Page 6 of 35
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`

`

`2
`
`Contents
`
`W. Wang / International Journal of Pharmaceutics 289 (2005) 1–30
`
`1.
`
`2.
`
`Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Protein aggregation and its influencing factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.1. Mechanisms of physical aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.1.1.
`Folding/unfolding intermediates and protein aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.1.2. Nucleation and growth of protein aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.1.3. Reversibility and specificity of physical aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.1.4. Thermodynamics and kinetics of protein aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.1.5. Computer-assisted probing of protein aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.2. Chemically-induced protein aggregation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.2.1. Disulfide bond formation/exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.2.2. Non-disulfide crosslinking pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.2.3. Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.2.4. Maillard reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Factors affecting protein aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.3.1.
`Primary structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.3.2.
`Secondary structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2.3.3. External factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`2.3.
`
`3. Characterization of protein aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.1. Morphology of protein aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.2.
`Structure and dissolution of protein aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`3.3. Analytical techniques in monitoring protein aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`4.
`
`Inhibition of protein aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.1. Refolding of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.1.1. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.1.2.
`Protein concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.1.3. Denaturant concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.1.4. Use of additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.1.5. Miscellaneous techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Processing of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.2.1. Heat treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.2.2.
`Shaking and shearing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.2.3.
`Freezing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.2.4. Drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4.2.5. Miscellaneous processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Storing of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`4.2.
`
`4.3.
`
`5.
`
`Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`2
`
`3
`3
`3
`4
`4
`5
`6
`6
`6
`6
`7
`7
`7
`7
`7
`9
`
`9
`10
`10
`11
`
`13
`13
`13
`18
`18
`19
`19
`19
`19
`20
`21
`21
`21
`22
`
`23
`
`24
`
`References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`24
`
`1. Introduction
`
`The past two decades saw an explosive growth
`in biopharmaceutics, fueled by the advancement of
`
`sensitive and high-throughput analytical methodolo-
`gies. Yet, a rapid commercialization of protein drug
`candidates has not been fully realized due to the
`presence of many road barriers. One undisputable
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`3
`
`barrier is the physical and chemical instabilities of
`proteins (Wang, 1999, 2000). Among these physical
`and chemical deterioration pathways, protein aggre-
`gation is arguably the most common and troubling
`manifestation of protein instability, almost
`in all
`phases of protein drug development. Presence of any
`insoluble aggregates in a protein pharmaceutical is
`generally unacceptable for product release.
`Protein aggregation is also well represented in
`human etiology. More than 20 different diseases are
`caused at least partially by abnormal protein aggre-
`gation (Stefani and Dobson, 2003), which may result
`from mutations and physical or chemical changes
`of cellular environment (Koo et al., 1999). Among
`these are Alzheimer’s disease, Parkinson disease,
`prion diseases (bovine spongiform encephalopathy
`and Creutzfeldt-Jacob diseases), Huntington’s disease,
`Down’s syndrome, cataract, and sickle cell disease.
`Some of these diseases are characterized by formation
`of protein fibrils, such as sickling cell disease (intra-
`cellular fibrillation of hemoglobin) and Alzheimer
`disease (extracellular fibrillation of amyloid ␤ peptide
`(A␤) (Koo et al., 1999). It is the protein aggregates
`(such as fibrils) that usually cause cytotoxicities
`(Stefani and Dobson, 2003).
`Although significant progress has been made,
`our current understanding of protein aggregation is
`still
`incomplete (Gupta et al., 1998; Carpenter et
`al., 1999; Chi et al., 2003). Its prevention or even
`moderate inhibition has been mostly experimental.
`Therefore, achieving a better understanding of protein
`aggregation is critical not only in various biophar-
`maceutical processes but also in finding a solution
`to those devastating diseases. This review article
`intends to discuss protein aggregation and its related
`mechanisms,
`to analyze factors affecting protein
`aggregation, and more importantly,
`to summarize
`possible venues in aggregation prevention/inhibition.
`It is not the intention of this paper to be exhaustive in
`literature review but to stimulate more intensive and
`vigorous investigation on protein aggregation.
`
`2. Protein aggregation and its influencing
`factors
`
`Protein molecules may aggregate simply by
`physical association with one another without any
`changes in primary structure (physical aggregation)
`
`or by formation of a new covalent bond(s) (chemical
`aggregation). Formation of such a bond(s) can either
`directly crosslink proteins (aggregation), or indirectly
`alter the aggregation tendency of the original protein
`(Finke et al., 2000). Both mechanisms can occur
`simultaneously to a protein and may lead to formation
`of either soluble or insoluble aggregates, depending
`on the protein, environmental condition, and stage
`of the aggregation process. For example, insulin can
`undergo both physical aggregation process, leading
`to formation of either soluble hexamers or insoluble
`fibrils and chemical aggregation process,
`leading
`to formation of either soluble dimmers via cyclic
`anhydride intermediate or insoluble disulfide-bonded
`aggregates (Sluzky et al., 1991, 1992; Costantino et
`al., 1994a, 1994b; Darrington and Anderson, 1995).
`Protein aggregation has been observed frequently
`in several key biopharmaceutical processes, including
`fermentation (Georgiou and Valax, 1999; Finke et al.,
`2000), refolding (van den Berg et al., 1999a, 1999b;
`Smith and Hall, 2001; Nguyen and Hall, 2002), shear-
`ing/shaking (Katakam et al., 1995), freeze-thawing
`(Kreilgaard et al., 1998a, 1998b), drying (Andya et
`al., 1999), reconstitution (Zhang et al., 1995a, 1995b),
`and storage (Vemuri et al., 1993; Costantino et al.,
`1994a, 1994b). The following section will discuss
`aggregation mechanisms and its influencing factors.
`
`2.1. Mechanisms of physical aggregation
`
`2.1.1. Folding/unfolding intermediates and
`protein aggregation
`A traditional view of protein aggregation is the asso-
`ciation of the unfolded state(s) of proteins. This view
`is supported by model predictions and experimental
`data as well (De Young et al., 1993; Stigter and Dill,
`1993). However, there is overwhelming evidence that
`protein folding/unfolding intermediates are precursors
`in protein aggregation (Fields et al., 1992; Fink, 1998),
`even though the intermediates are usually not stable
`and poorly populated (Murphy et al., 1992). In contrast,
`completely folded or unfolded proteins do not aggre-
`gate easily as the hydrophobic side chains are either
`mostly buried out of contact with water, or randomly
`scattered (Uversky et al., 1999). It is the patches of con-
`tiguous hydrophobic groups in the folding/unfolding
`intermediates that initiate the aggregation process. Ag-
`gregation of many proteins has been shown to be initi-
`
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`
`ated by intermediates, such as scrapie amyloid (prion)
`protein (PrP27–30) (Safar et al., 1994), carbonic an-
`hydrase B (CAB) (Cleland and Wang, 1992; Ham-
`marstrom et al., 1999), recombinant human growth hor-
`mone (rhGH) (Bam et al., 1996), insulin (Brange et al.,
`1997), human lysozyme variants (Booth et al., 1997),
`P22 tailspike polypeptide (Speed et al., 1997; Schuler
`et al., 1999), and phage P22 wild-type coat protein and
`its mutants (A108V, G232D, F353L) (Teschke, 1999).
`Computer simulation studies also demonstrate origi-
`nation of the aggregation process primarily from in-
`teractions of partially folded intermediates (Gupta et
`al., 1998; Istrail et al., 1999). Thermal treatment can
`easily generate protein-unfolding intermediates, which
`can rapidly aggregate, such as ovalbumin (Kato and
`Takagi, 1988), and a mutant of Cro repressor (Fabian
`et al., 1999). Another factor contributing to the rapid
`aggregation of intermediates is their high rate of diffu-
`sion relative to the folded state (Damodaran and Song,
`1988). The high diffusion rate can significantly increase
`the chance of association of the intermediates.
`The intermediates can be formed either from the
`folded or unfolded state (Wetzel, 1996). In many cases,
`more than one intermediate may exist, such as CAB
`(Wetlaufer and Xie, 1995), staphylococcal nuclease
`(Uversky et al., 1999), a mutant of Cro repressor
`(Fabian et al., 1999), and immunoglobulin G (IgG)
`(Vermeer and Norde, 2000). In addition, the number
`and structure of folding intermediates may be different
`from those of unfolding intermediates (Kim and Yu,
`1996). Since the folding intermediates can be formed
`locally or globally, these intermediates may have sig-
`nificant amount of secondary structures, and even ter-
`tiary structures (Safar et al., 1994; Kendrick et al.,
`1998a, 1998b; Uversky et al., 1999).
`
`2.1.2. Nucleation and growth of protein
`aggregates
`Based on the above analysis, the aggregation process
`can be described as scheme (1), where proteins form
`reversible unfolding intermediates, which then form re-
`versible unfolded proteins or irreversible/reversible ag-
`gregates.
`
`to the
`The intermediate state (I) is equivalent
`aggregation-competent state (A) or transition state
`(TS*) as proposed recently by other investigators
`(Krishnamurthy and Manning, 2002; Chi et al., 2003).
`The process from N to A can be considered as the nu-
`cleation step, which is usually rate limiting; in another
`word, the aggregation process is nucleation dependent.
`Nucleation has been demonstrated or suggested to be
`the initial step for fibrillation of a sequence of E. coli.
`OsmB protein (Jarrett and Lansbury, 1993), aggrega-
`tion of K97I interleukin 1␤ during refolding (Finke
`et al., 2000), and aggregation of ␤-amyloid peptide
`(Lomakin et al., 1997; Szabo et al., 1999).
`Further growth of protein aggregates after nucle-
`ation can be divided into two types: monomer–cluster
`aggregation (addition of a monomer to a growing mul-
`timer) and cluster–cluster aggregation (addition of a
`multimer to another multimer) (Speed et al., 1997).
`These two processes can be described as schemes (2)
`and (3) (Patro and Przybycien, 1994).
`nI + Am → (n − 1)I + Am+1
`Am + An → Am+n
`where Am and An are aggregates composed of m and n
`monomers. These two processes can occur at the same
`time to the same protein. Examples of the first type of
`aggregation include staphylococcal nuclease (Uversky
`et al., 1999), and amyloid formation (Tomski and
`Murphy, 1992; Lomakin et al., 1997). Cluster–cluster
`polymerization was responsible for the aggregation of
`P22 tailspike polypeptide (a trimer) chains during in
`vitro refolding (Speed et al., 1997). The initial protein
`aggregates are soluble but gradually become insoluble
`as they exceed certain size and solubility limits (Fink,
`1998; Uversky et al., 1999). Such examples include ag-
`gregation of CAB during refolding (Cleland and Wang,
`1990) and aggregation of human serum albumin (HSA)
`◦
`during storage at 40, 55 or 70
`C (Oliva et al., 1999).
`
`(2)
`
`(3)
`
`2.1.3. Reversibility and specificity of physical
`aggregation
`The reversibility of protein aggregation is usually
`dependent on the stage of the aggregation process.
`The initial formation of soluble aggregates (nucle-
`ation) may be reversible but the later formation of in-
`soluble aggregates is usually irreversible, unless pre-
`cipitation is artificially induced such as during salt-
`
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`
`ing out. The two different stages correspond, res