Current opinion in biotechnology
`DUP - Cener=il Collectiori
`VV1 CU7::,JG8l.
`v 12 no ~
`Apr ~·00 1
`
`e onnewald
`
`a·
`td!0th8mical engineering
`ed by B.
`nan D Kelley
`
`PROPERTY OF THE
`NATIONAL
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`I online with :·io,,1~ecfNet Reviews
`. 'lltcommend access to yourhlibrlllian~~,~d
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`1 of 11
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`Fresenius Kabi
`Exhibit 1016
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`Current Opinion in Biotechnology
`Editors Martin Rosenberg USA Kenneth Timmis Germany
`
`Vol 1 2 No 2 April 2001
`
`Editorial Board
`
`2001 Contents
`
`The subject of biotechnology is divided into 10
`major sections, each of which is reviewed once a
`year. Each issue contains one or two of the major
`sections, and the amount of space devoted to each
`section is related to its importance.
`
`February
`Analytical biotechnology
`Edited by Jorg D Hoheisel and Patricia J Conway
`
`April
`Biochemical engineering
`Edited by Brian D Kelley
`Plant biotechnology
`Edited by Uwe Sonnewald
`
`June
`Environmental biotechnology
`Edited by Shigeaki Harayama
`Regulatory affairs
`Edited by Kathryn Zoon
`
`August
`Protein technologies and commercial
`enzymes
`Edited by Lutz Jermutus and Joelle Pelletier
`
`October
`Expression vectors and delivery systems
`Edited by Hansji:irg Hauser and Gerben Zylstra
`Food biotechnology
`Edited by Beat Mollet
`
`December
`Chemical biotechnology
`Edited by Robert Azerad
`Pharmaceutical biotechnology
`Edited by Stanley Crooke
`
`\
`
`James E Bailey (Switzerland)
`Frederick Brown (USAI
`Mark F Cantley (Belgium)
`Jean-Claude Chermann (France)
`Nam-Hai Chua (USA)
`Julian Davies (Canada)
`Tim JR Harris IusAI
`Lee Hood (USAI
`Koki Horikoshi (Japan)
`C Richard Hutchinson IUSAI
`Massayori Inouye (USAI
`lsao Karu be (Japan)
`Sung-Hou Kim (USAI
`Christopher R Lowe (UKI
`Sheldon W May IusAI
`Joachim Messing IusAI
`Klaus Mosbach (Sweden)
`Bernard Moss IusAI
`Robin Offord (Switzerland)
`George Paste (USAI
`Eugene Rosenberg (Israel)
`Jeffrey Schell (Germany!
`Allan R Shatzman (USAI
`Graham Strachan (Canada)
`Arthur D Strosberg (France!
`Mathias Uhlen (Sweden)
`Daniel Wang (USAI
`Alan Williamson (USA)
`
`Th is ma t erial w as co.,ied
`at t he N LM and may t,e
`Subje<t US Co pyright Laws
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`Current Opinion in Biotechnology
`Contents
`
`Vol 12 No 2 April 2001
`
`113
`
`Paper alert
`
`121
`
`Web alert
`
`Reviews
`
`Plant biotechnology
`Edited by Uwe Sonnewald
`
`123
`
`126
`
`131
`
`135
`
`139
`
`144
`
`150
`
`155
`
`161
`
`Uwe Sonnewald
`Editorial overview
`Light at the end of the tunnel: from genes to function
`
`Yangrae Cho and Virginia Wal bot
`Computational methods for gene annotation: the Arabidopsis
`genome
`
`Michel Rossignol
`Analysis of the plant proteome
`
`Richard N Trethewey
`Gene discovery via metabolic profiling
`
`Barbara Hohn, Avraham A Levy and Holger Puchta
`Elimination of selection markers from transgenic plants
`
`Aart JE van Bel, Julian Hibberd, Dirk Priifer and
`Michael Knoblauch
`Novel approach in plastid transformation
`
`Wan Xiang Li and Shou Wei Ding
`Viral suppressors of RNA silencing
`
`Gert Forkmann and Stefan Martens
`Metabolic engineering and applications of flavonoids
`
`Rudiger Hell and Helke Hillebrand
`Plant concepts for mineral acquisition and allocation
`
`~<:>mmentary
`
`169
`
`Thomas J Oh and Gregory D May
`Oligonucleotide-directed plant gene targeting
`
`Biochemical engineering
`Edited by Brian D Kelley
`
`180
`
`188
`
`195
`
`202
`
`208
`
`212
`
`Lily Chu and David K Robinson
`Industrial choices for protein production by large-scale cell culture
`
`Helen E Chadd and Steven M Chamow
`Therapeutic antibody expression technology
`
`James R Swartz
`Advances in Escherichia coli production of therapeutic proteins
`
`Eliana De Bernardez Clark
`Protein refolding for industrial processes
`
`Robert van Reis and Andrew Zydney
`Membrane separations in biotechnology
`
`Jeffrey L Cleland, Ann Daugherty and Randall Mrsny
`Emerging protein delivery methods
`
`Product news
`
`The cover
`Antibody therapeutics can potentially treat diseases ranging from
`autoimmune disorders to cancer and viral or bacterial infections. The
`emergence of antibodies as an attractive therapy is the result of the
`evolution of monoclonal antibody technology from 100% mouse
`protein through chimeric and humanized proteins to fully human
`antibodies. The figure shows chimeric (67% human), humanized
`(90-95% human) and human antibodies. Mouse-derived sequences
`are shown in blue and human-derived sequences in purple. See Chadd
`and Chamow (pp 188-194) for a review on therapeutic antibody
`expression technology.
`
`How to claim your FREE on line access to
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`173
`
`175
`
`Brian D Kelley
`Editorial overview
`Bioprocessing of therapeutic proteins
`
`Tip: If you do not use a shared terminal, you can tick tho 'save pass(cid:173)
`word' box when you first log on to BioMedNet so that you only need
`to log on once.
`
`Haley A Laken and Mark W Leonard
`Understanding and modulating apoptosis in industrial cell culture
`
`If you have any questions e-mail: info@current-tronds.com
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`Th is ma t erial was copied
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`5'ubject US Copyright Laws
`
`Continued
`
`4 of 11
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`

`Vol 12 No 2 April 2001
`
`The next issue of this journal
`
`Environmental biotechnology
`Edited by Shigeaki Harayama
`
`Will contain reviews by
`
`Kazuya Watanabe
`Microorganisms relevant to bioremediation
`
`Rudolf Amann, Bernhard M Fuchs and Sebastian Behrens
`In situ identification of microorganisms by fluorescence in situ hybridisation
`
`Masayuki Shimao
`Biodegradation of plastics
`
`Ultan F Walsh, John P Morrissey and Fergal O'Gara
`Pseudomonas for biocontrol of phytopathogens: from functional
`genomics to commercial exploitation
`
`Mike SM Jetten, Michael Wagner, John Fuerst, Mark van
`Loosdrecht, Gijs Kuenen and Marc Strous
`Microbiology and application of the anaerobic ammonium oxidation
`'anammox' process
`
`Dick B Janssen, Jantien E Oppentocht and Gerrit J Poelarends
`Microbial dehalogenation
`
`Regulatory affairs
`Edited by Kathryn Zoon
`
`Will contain reviews by
`
`Herbert A Smith and Dennis M Klinman
`The regulation of DNA vaccines
`
`Eda T Bloom
`Xenotransplantation: regulatory challenges
`
`Jonathan R Lloyd and Derek R Lovley
`Microbial detoxification of metals and radionuclides
`
`Kathryn E Stein and Keith O Webber
`The regulation of biologic products derived from bioengineered plants
`
`Yuji Sekiguchi, Yoichi Kamagata and Hideki Harada
`Recent advances in methane fermentation technology
`
`Anthony S Lubiniecki and John C Petricciani
`Recent trends in cell substrate considerations for continuous cell lines
`
`Other Current articles of interest to readers of
`this issue
`Reviews
`
`Protein expression in plastids by Peter B Heifetz and Anne Marie
`Tuttle. Current Opinion in Plant Biology 2001, 4:157-161.
`
`Sequence and analysis of the Arabidopsis genome by Michael
`Bevan, Klaus Mayer, Owen White, Jonathan A Eisen, Daphne Preuss,
`Thomas Bureau, Steven L Salzberg and Hans-Werner Mewes.
`Current Opinion in Plant Biology 2001, 4:105-11 O.
`
`Cytochrome P450s as genes for crop improvement by Kenneth A
`Feldmann. Current Opinion in Plant Biology 2001, 4:162-167.
`
`Computational methods for protein function analysis by Matteo
`Pellegrini. Current Opinion in Chemical Biology 2001, 5:46-50.
`
`Mechanisms of protein folding by Viara Grantcharova, Eric J Alm,
`David Baker and Arthur L Horwich. Current Opinion in Structural
`Biology 2001, 11 :70-82.
`
`Gene silencing and DNA methylation processes by Jerzy
`Paszkowski and Steven A Whitham. Current Opinion in Plant
`Biology 2001, 4:123-129.
`
`All these articles are also available online in the
`BioMedNet library
`http://bmn.com
`
`Th is mate rial wasrnpcied
`at the NLM and may bi=
`~u bje<t US Copcyright Laws
`
`5 of 11
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`202
`
`Protein refolding for industrial processes
`Eliana De Bernardez Clark
`
`Inclusion body refolding processes are poised to play a major
`role in the production of recombinant proteins. Improving
`renaturation yields by minimizing aggregation and reducing
`chemical costs are key to the industrial implementation of
`these processes. Recent developments include solubilization
`methods that do not rely on high denaturant concentrations
`and the use of high hydrostatic pressure for simultaneous
`solubilization and renaturation.
`
`Addresses
`Department of Chemical and Biological Engineering, Tufts University,
`Medford, MA 02155, USA; e-mail: eliana.clark@tufts.edu
`
`Current Opinion in Biotechnology 2001, 12:202–207
`
`0958-1669/01/$ — see front matter
`© 2001 Elsevier Science Ltd. All rights reserved.
`
`Abbreviations
`CTAB
`n-cetyl trimethylammonium bromide
`DTE
`dithioerythritol
`DTT
`dithiothreitol
`GdmCl guanidinium chloride
`PDGF
`platelet-derived growth factor
`SDS
`sodium dodecyl sulfate
`SEC
`size-exclusion chromatography
`
`Introduction
`The need for the efficient production of genetically engi-
`neered proteins has grown and will continue to grow as a
`consequence of the success of the human genome project. A
`variety of hosts may be used to produce these proteins, with
`expression in bacteria poised to play a major role, particular-
`ly when the biological activity of the protein product is not
`dependent on post-translational modifications. Expression
`of genetically engineered proteins in bacteria often results
`in the accumulation of the protein product in inactive insol-
`uble deposits inside the cells, called inclusion bodies. Faced
`with the prospect of producing an insoluble and inactive
`protein, researchers usually attempt to improve solubility by
`a variety of means, such as growing the cells at lower tem-
`peratures, co-expressing the protein of interest with
`chaperones and foldases and using solubilizing fusion part-
`ners, among others [1]. However, expressing a protein in
`inclusion body form can be advantageous. Large amounts of
`highly enriched proteins can be expressed as inclusion bod-
`ies. Trapped in insoluble aggregates, these proteins are for
`the most part protected from proteolytic degradation. If the
`protein of interest is toxic or lethal to the host cell, then
`inclusion body expression may be the best available pro-
`duction method. The challenge is to take advantage of the
`high expression levels of inclusion body proteins by being
`able to convert inactive and misfolded inclusion body
`proteins into soluble bioactive products [2–5].
`
`The recent literature includes many examples of the
`refolding of genetically engineered proteins. A significant
`
`number of these publications deal with the expression
`and purification of small amounts of proteins for structure/
`function relationship and biophysical characterization
`studies. Although valuable, the processes described in
`these publications are usually inefficient, include multiple
`unnecessary steps and have very low recovery yields. A
`second significant fraction of the refolding literature deals
`with understanding the folding pathway of a variety of pro-
`teins and, in particular, early folding events. These studies
`are performed with purified proteins that are subjected to
`unfolding under a variety of conditions, followed by
`carefully designed and monitored refolding experiments.
`A third fraction of the refolding literature, and the focus of
`this review, deals with the development of more efficient
`refolding methods that can be used for the commercial
`production of genetically engineered proteins
`
`The general strategy used to recover active protein from
`inclusion bodies involves three steps: inclusion body isola-
`tion and washing; solubilization of the aggregated protein;
`and refolding of the solubilized protein (Figure 1a).
`Although the efficiency of the first two steps can be rela-
`tively high, renaturation yields may be limited by the
`accumulation of inactive misfolded species as well as aggre-
`gates. Because the majority of industrially relevant proteins
`contain one or more disulfide bonds, this review focuses on
`recent advances in oxidative protein refolding, that is,
`refolding with concomitant disulfide-bond formation.
`
`Inclusion body isolation, purification and
`solubilization
`Inclusion bodies are dense, amorphous protein deposits that
`can be found in both the cytoplasmic and periplasmic space
`of bacteria [1,6•]. Structural characterization studies using
`ATR-FTIR (attenuated total reflectance Fourier-trans-
`formed infrared spectroscopy) have shown that the
`insoluble nature of inclusion bodies may be due to their
`increased levels of non-native intermolecular β-sheet con-
`tent compared with native and salt-precipitated protein
`[7,8]. Cells containing inclusion bodies are usually disrupted
`by high-pressure homogenization or a combination of
`mechanical, chemical and enzymatic methods [6•,9•]. The
`resulting suspension is treated by either low-speed centrifu-
`gation or filtration to remove soluble proteins from the
`particulate containing the inclusion bodies. The most
`difficult to remove contaminants of inclusion body protein
`preparations are membrane-associated proteins that are
`released upon cell breakage. Washing steps are performed to
`remove membrane proteins and other contaminants.
`Methods used to solubilize prokaryotic membrane proteins
`can be adapted to wash inclusion bodies. The most common
`washing steps utilize EDTA, and low concentrations of
`denaturants and/or weak detergents such as Triton X-100,
`deoxycholate and octylglucoside [6•,9•,10,11•,12,13,P1,P2].
`
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`Batas, Schiraldi and Chaudhuri [10] recently compared
`centrifugation and membrane filtration for the recovery
`and washing of inclusion bodies. Two membrane pore sizes
`(0.1 and 0.45 μm) were compared; the larger pore size
`membrane gave better solvent flux and protein purity.
`Centrifugation resulted in higher protein purity, probably
`because it takes advantage of the density differences
`between cell debris and inclusion bodies.
`
`A variety of methods may be used to solubilize inclusion
`bodies; however, the choice of solubilizing agent can great-
`ly impact the subsequent refolding step and the cost of the
`overall process. The most commonly used solubilizing
`agents are denaturants, such as guanidinium chloride
`(GdmCl) and urea. Using these denaturants, solubilization
`may be accomplished by the complete disruption of the
`protein structure (unfolding) or by the disruption of inter-
`molecular interactions with partial unfolding of the
`protein. The latter approach has the advantage that it
`requires lower amounts of denaturant to succeed.
`Although proteins have been successfully refolded from
`the denatured state, it may prove to be difficult to fold pro-
`teins from a partially folded state. Key to the development
`of an efficient and economic denaturant-based solubiliza-
`tion step is the determination of the minimum amount of
`denaturant needed to solubilize the protein and to allow
`for full bioactivity recovery in the refolding step. The
`majority of the published work on inclusion body protein
`refolding has used relatively high denaturant (6–8 M) and
`protein (1–10 mg/ml) concentrations in the solubilization
`step [5,9•,10,11•,12–14].
`
`Lower denaturant concentrations (1–2 M) have been used
`to solubilize cytokines from Escherichia coli inclusion bod-
`ies [P3]. The purity of the solubilized protein was much
`higher at GdmCl concentrations of 1.5–2 M compared with
`the more commonly used 4–6 M concentrations, because
`at the higher GdmCl concentrations contaminating pro-
`teins were also released from the particulate fraction. No
`information was provided about the efficiency of this solu-
`bilization process or the range of inclusion body protein
`concentrations for successful solubilization.
`
`Extremes of pH have also been used to solubilize inclusion
`bodies. Gavit and Better [15] used a combination of low pH
`(≤ 2.6) and high temperature (85°C) to solubilize antifungal
`recombinant peptides from E. coli. Lower temperatures and
`higher pH values resulted in increased solubilization time.
`Reddy and coworkers [16] utilized 20% acetic acid to solu-
`bilize a maltose-binding protein fusion from inclusion
`bodies. These low pH solubilization processes may not be
`applicable to many proteins, particularly those that undergo
`irreversible chemical modifications at these conditions or
`those susceptible to acid cleavage.
`
`High pH (≥ 12) has been used to solubilize growth hormones
`[17,18] and proinsulin [P4]. Exposure to elevated pH condi-
`tions for extended periods of time may also cause irreversible
`
`Protein refolding for industrial processes De Bernardez Clark 203
`
`Figure 1
`
`(a) Cells containing inclusion bodies
`
`Homogenization
`Centrifugation or microfiltration
`
`Soluble fraction
`
`Particulate
`Washing
`Solubilization
`Centrifugation or microfiltration
`
`Soluble proteins
`
`Debris
`
`Renaturation with or without
`prior purification
`
`Active protein
`
`(b) Cells containing inclusion bodies
`
`In situ solubilization
`Centrifugation or microfiltration
`
`Soluble fraction
`
`Debris
`
`Purification
`Renaturation
`
`Active protein
`
`Current Opinion in Biotechnology
`
`Processes for the recovery of inclusion body proteins. (a) Inclusion
`body isolation followed by solubilization. (b) The in situ solubilization of
`inclusion bodies.
`
`chemical modifications to the protein. Thus, this high pH
`solubilization method, although attractive for its simplicity
`and low cost, may not be applicable to most pharmaceutical
`proteins. More effective solubilization methods for growth
`hormones combine high pH with low denaturant concentra-
`tions [17,18], 20–40% isopropyl or n-propyl alcohol solutions
`[P1] or acyl glutamate detergents [P5].
`
`Detergents have also been used to solubilize inclusion bodies.
`Commonly used detergents are sodium dodecyl sulfate
`(SDS) and n-cetyl trimethylammonium bromide (CTAB)
`[3,18,19]. Detergents offer the advantage that the solubilized
`protein may already display biological activity, thus avoiding
`the need for a refolding step. If this is the case, it is important
`to remove contaminating membrane-associated proteases in
`the inclusion body washing step to avoid proteolytic degrada-
`tion of the solubilized inclusion body protein [6•]. One
`acknowledged drawback of the use of detergents as solubiliz-
`ing agents is that they may interfere with downstream
`chromatographic steps. Extensive washing may be needed to
`remove solubilizing detergents [P5]. Alternatively, detergents
`may be extracted from refolding mixtures by using cyclodex-
`trins [20], linear dextrins [21] or cycloamylose [22].
`
`7 of 11
`
`Fresenius Kabi
`Exhibit 1016
`
`

`

`204 Biochemical engineering
`
`Patra and coworkers [18] compared several solubilization
`methods for the recovery of human growth hormone from
`E. coli inclusion bodies. They observed similar solubiliza-
`tion efficiencies when using 8 M urea, 6 M GdmCl, 1%
`SDS or 1% CTAB (all at pH 8.5) or 2 M urea (at pH 12.5).
`Refolding for the first four solubilization conditions
`required a dilution step resulting in increased process vol-
`umes. Solubilization in 2 M urea at pH 12.5 was simple,
`economical and efficient, and refolding could be accom-
`plished by a simple pH adjustment without dilution.
`However, this high pH solubilization method may not be
`applicable to proteins that might undergo irreversible
`chemical modifications under these conditions.
`
`A key to the solubilization process is the addition of a
`reducing agent to maintain cysteine residues in the
`reduced state and thus prevent non-native intra- and inter-
`disulfide bond formation in highly concentrated protein
`solutions at alkaline pH. Typically used reducing agents
`are dithiothreitol (DTT), dithioerythritol (DTE), and
`2-mercaptoethanol [2,3]. These reducing agents should be
`added in slight excess to ensure complete reduction of all
`cysteine residues. Chelating agents are added to the solu-
`bilization solution to prevent metal-catalyzed air oxidation
`of cysteines. Alternatively, reduced cysteines may be pro-
`tected from oxidation by the formation of S-sulfonate
`derivatives [23,P6,P7] or mixed disulfides [9•,P7].
`
`When expression levels are very high, a competitive alter-
`native is to add the solubilizing agents directly to the broth
`at the end of the fermentation process. This in situ solubi-
`lization method has been used to recover insulin-like
`growth factor using urea under alkaline conditions [P8] and
`antifungal recombinant peptides using a combination of
`low pH (< 2.6) and high temperature (85°C) [15]. The
`main disadvantage of in situ solubilization concerns the
`release of both proteinaceous and nonproteinaceous conta-
`minants that may have to be removed before renaturation
`is attempted. It has been shown that protein refolding in
`the presence of impurities may result in decreased yields
`[6•,24]. The main advantage of this method is the elimina-
`tion of time-consuming and energy-consuming mechanical
`disruption methods and of one centrifugation and/or
`filtration step (Figure 1b).
`
`Solubilization may also be accomplished by applying high
`hydrostatic pressures (1–2 kbar) in the presence of reducing
`agents and low concentrations of solubilizing agents [25•,P9].
`
`Renaturation of the solubilized protein
`When inclusion bodies have been solubilized using a com-
`bination of reducing agents and high concentrations of
`denaturants, renaturation is then accomplished by the
`removal of excess denaturants by either dilution or a
`buffer-exchange step, such as dialysis, diafiltration, gel-fil-
`tration chromatography or immobilization onto a solid
`support. Because of its simplicity, dilution of the solubi-
`lized protein directly into renaturation buffer is the most
`
`commonly used method in small-scale refolding studies.
`The main disadvantages of dilution refolding for commer-
`cial applications are the need for larger refolding vessels
`and additional concentration steps after renaturation. The
`key to successful dilution refolding is to control the rate of
`the addition of denatured protein to renaturation buffer
`and to provide good mixing in order to maintain low pro-
`tein concentration during refolding and thus prevent
`aggregation. Dilution refolding can also be accomplished
`in multiple steps, also known as ‘pulse renaturation’, in
`which aliquots of denatured reduced protein are added to
`renaturation buffer at successive time intervals [2,9•], or
`semicontinuously via fed-batch addition of the denatured
`reduced protein to refolding buffer [26]. Recently, Katoh
`and Katoh [26] developed a continuous refolding method
`in which denatured reduced protein is gradually added
`from the annular space of a membrane tube to renaturation
`buffer flowing continuously through the inner space of the
`membrane tube. Refolding yields obtained using this con-
`tinuous refolding method were similar to those obtained
`using fed-batch dilution and about 10% higher than those
`using batch dilution [2