Journal of Biotechnology 128 (2007) 587–596

Review

Current status of technical protein refolding

Alois Jungbauer ∗, Waltraud Kaar 1

Department of Biotechnology, Austrian Center of Biopharmaceutical Technology, University of Natural Resourcesand Applied Life Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria

Received 30 July 2006; received in revised form 14 November 2006; accepted 4 December 2006

Abstract

The expression of heterologous proteins in microbial hosts frequently leads to the formation of insoluble aggregates. Tofully exploit the production capacity of the cells, efficient strategies for further processing have to be developed. While in labscale matrix assisted refolding techniques, especially of histidine-tagged proteins have become very popular, in production scalerefolding by dilution is still predominant due to its simplicity. However scaling up dilution processes leads to large volumes andlow protein concentration. This is a heavy burden both for liquid handling and for subsequent downstream processing steps.Process development aims to operate at uniform, reproducible conditions, to reduce costs to a minimum and to guarantee therequired quality of the product. The general refolding kinetics, exploration of appropriate refolding conditions are reviewed.The major refolding operations such as dilution, matrix assisted refolding, pressure driven refolding or continuous refolding

applications are discussed in view of industrial applicability.© 2006 Elsevier B.V. All rights reserved.

Keywords: Inclusion bodies; Escherichia coli; Refolding; Large scale

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5882. Refolding kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588

3. Isolation of recombinant protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5904. Determination of refolding conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5915. Refolding by dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5926. Pressure treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592

∗ Corresponding author. Fax: +43 1 3697615.E-mail address: alois.jungbauer@boku.ac.at (A. Jungbauer).

1 Current address: Centre for Biomolecular Engineering, Australian Institute for Bioengineering and Nanotechnology, University ofQueensland, St. Lucia 4072, Qld, Australia.

0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jbiotec.2006.12.004

8. Analysis of folded proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5939. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594

. . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Recombinant DNA technology allows the expres-ion of valuable heterologous proteins at highxpression rates. Particularly in Escherichia coli (E.oli) overexpression of proteins often leads to aggrega-ion and deposition in dense, insoluble particles withinhe host cell, so-called inclusion bodies (IB). Theyre easily distinguishable from other cell componentsue to their refractile character (Fig. 1). Formation ofnclusion bodies is heavily protein dependent, chargeistribution and turn forming residues have a strongmpact (Wilkinson and Harrison, 1991), also presencef cysteines may enforce tendency of aggregate forma-ion (Rinas et al., 1992) but it may also be influencedy altering cell cultivation conditions (Panda et al.,999). Decelerated cell growth achieved by lower tem-erature (Schein and Noteborn, 1988) or suboptimalH (Kopetzki et al., 1989) can result in the produc-ion of soluble recombinant proteins which indicates

hat the cells are overburdened by the protein produc-ion at regular cultivation conditions. Still productionf proteins as inclusion bodies is favored in severalases. Intracellular expression of proteins does have

ig. 1. Electron micrograph of E. coli cells containing cytosolicnclusion bodies.

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s

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594

ertain advantages over secretion of the product intohe culture supernatant. Design heuristics of biotech-ological processes recommend removing the mostbundant impurities first. In a fermentation processhis constitutes water. By a simple unit operation suchs centrifugation the product can be concentrated byecovering the whole cells in the sediment. As outlinedn Fig. 2 the attraction of inclusion body productionompared to secretory systems is the simple primaryecovery step. After cell harvest the cells have to beisintegrated and inclusion bodies have to be separatedrom cell debris and soluble cell components releasednto the homogenate.

Inclusion bodies consist nearly exclusively ofecombinant proteins (Speed et al., 1996). Isolation ofhe desired product at already high purity is relativelyasy due to density differences (Schoner et al., 1985)nd high protein concentration can be achieved at therimary solubilization step. Although there are studies,hat inclusion body protein is not the dead end deposits believed earlier but inclusion bodies are dynamictructures subjected to permanent conversion (Carriond Villaverde, 2002), the storage as aggregates stilleatures distinct protection from protease degradationCheng et al., 1981; Kitano et al., 1987). Anotherdvantage is the possibility to produce compounds,hich are cell toxic in higher concentration. However,

s a major disadvantage, the subsequently requiredefolding procedure poses a bottleneck in everyownstream scheme. Protein aggregates have toe resolved and folded into their native structure.arious strategies have been employed to achieve anctive compound refolded from inclusion bodies ineasonable yield. In this review special emphasis isaken on the scalability of a method and the use inndustrial production processes.

588 A. Jungbauer, W. Kaar / Journal of Biotechnology 128 (2007) 587–596

7. Large scale chromatographic refolding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593

. Refolding kinetics

The distinct folding pathway of a single protein istill case of many hypotheses. While debating the driv-

A. Jungbauer, W. Kaar / Journal of Biotechnology 128 (2007) 587–596 589

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ng force of the folding process they agree in the facthat a protein undergoes different more or less unstableonformations until it reaches its final native structure.t the absence of chaotropic agents these intermediatesay exhibit intermolecular interactions, which leads

o the major problems in a refolding procedure, aggre-ation and precipitation of the proteins. In refoldingodels estimating the final yield of a renaturation

rocess, these competing side reactions are considereds of higher order while the folding reaction itself ispproximated by a first order reaction (Zettlmeisslt al., 1979). A refolding reaction may therefore beescribed as

dU

dt= −(k1U + k2NUn) (1)

ith k1 the net rate constant of folding, k2 the netate constant of aggregation, U the concentration ofnfolded protein, t time, N aggregation number and nhe reaction order of aggregation, assuming that backeaction from folded or aggregated protein to unfolded

rotein is negligible and formation of possible foldingntermediates is infinitely fast. Analytical solutions ofhis differential equation exist for second (Kiefhabert al., 1991) and third order aggregation reactionsHevehan and De Bernardez Clark, 1997) and areepicted in Eqs. (2) and (3), respectively.

ocibcd

lation from fermentation broth. Bold lines depict a usual inclusion

(t) = k1

U0K2ln

[1 + U0K2

k1(1 − e−k1t)

](2)

here Y(t) is the yield of the refolding reaction, U0 thenitial concentration of the denatured protein and K2 ishe apparent rate constant of aggregation, combiningggregation number and rate constant of aggregationn k2N.

(t) = Ψ{

tan−1 [(1 + Ψ2) e2k2t − 1]1/2 − tan−1 Ψ

}(3)

ith Ψ = (k1/k2U20 )

1/2, where k1 again is the net rate

onstant of folding, k2 net rate constant of aggregationnd U0 is the initial concentration of unfolded protein.

Common refolding techniques aim to inhibit theseide reactions to enhance the final yield of correctlyolded protein. Major attention has to be drawn tohe chemical as well as physical environment during aefolding process since folding and aggregation kinet-cs are heavily influenced thereby. Kinetic constants ofrefolding process are of importance for the design ofperation parameters such as dilution rate, final proteinoncentration and refolding time. Once optimal refold-

ng conditions are found, yield would be solely definedy protein concentration if an ideal dilution processould be applied, since aggregation is a concentrationriven process. The determination of kinetic constants

590 A. Jungbauer, W. Kaar / Journal of Biot

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ig. 3. Time course of the formation of native protein and aggregatesuring a refolding reaction.

ay be accomplished by a direct fit of an appropri-te refolding model such as shown in Eqs. (2) and (3)o data from dilution experiments at different proteinoncentration collected over refolding time until thendpoint is reached, or by an iterative approach whennly data for one concentration are available over thehole time range, while endpoint data are available

or different protein concentrations (Fig. 3). For a sec-nd order aggregation reaction yield at infinite time isescribed by

= k1

U0K2ln

(1 + U0K2

k1

), (4)

herefore kinetic constants can be easily extracted fromhe data set. However, kinetic constants heavily dependn refolding conditions and have to be determined forvery buffer to be used.

The required scale of a refolding process influenceshoice of a distinct methodology.

. Isolation of recombinant protein

While in lab scale cell lysis is often performednzymatically yielding in almost complete degradationf cell walls, the industrial means of inclusion bodysolation is mechanical disruption of cells followed by

entrifugation. After high pressure homogenization,hich results in a suspension of cell debris and theroduct, sedimentation of inclusion bodies has to beerformed. The design of a centrifugal process requires

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echnology 128 (2007) 587–596

nowledge of size distribution and density of the par-icles to be separated (Taylor et al., 1986). Monitoringeparation in disc stack centrifuges based on differentpectrophotometric properties of inclusion bodies andell debris (Jin et al., 1994) supports centrifugal processesign. Separation may prove critical if cell fragmentsnd inclusion bodies have similar sedimentation prop-rties. Pressure treatment for cell disintegration has toe optimized as has been studied in detail by Wongt al. (1997). They showed that repeated homogenizerasses resulted in better fractionation of inclusionodies and cell debris leading to increased inclusionody purity. As a result of cell breakage, outer mem-rane components are released and may get adsorbedo the inclusion body surface (Hart et al., 1990). Theseompounds can be removed by several detergentashing steps, however, detergents sometimes causeroblems in subsequent downstream processingnd are therefore possibly avoided (Choe et al.,006).

Chemical extraction of inclusion body protein asn alternative to mechanical means poses both advan-ages and disadvantages. Extraction directly fromermentation broths shortcuts unit operation steps,owever release of high molecular weight DNA leadso increased viscosity of the solution which mayause severe problems for the capture of the product.dditionally high amounts of host cell proteins are con-

ained in the extract. These problems were partly solvedn different approaches. Removal of DNA could bechieved by precipitation with spermine (Choe et al.,002) and also cheaper DNA-precipitants with com-arable efficacy are available (Choe et al., 2006). Aethod for the selective extraction of recombinant pro-

eins produced as inclusion bodies was described byalconer et al. (1999) and successfully transferred toilot scale. In a first step, membranes were permeabi-ized with a combination of urea and EDTA and hostell proteins were extracted, while inclusion bodiesere kept insoluble by surface oxidation with the helpf a disulfide bond promoting reagent. After removalf extracted compounds by diafiltration using a mem-rane with a high cut-off value, inclusion body proteinas solubilized under chaotropic and reducing condi-

ions. Compared with conventional extraction methodsonsisting of mechanical cell disruption, centrifugationteps and solubilization of inclusion bodies, similarrotein extraction and purity could be reached. As a

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A. Jungbauer, W. Kaar / Journal

rawback this method is only feasible for proteins withhigh cysteine content. In a different approach it was

imed to disintegrate cells while maintaining inclusionodies insoluble by treatment with a combination ofriton X-100 and EDTA (Lee et al., 2004). This methodhould be applicable for a wider range of recombinantroteins and might constitute a considerable shortcutn processing.

. Determination of refolding conditions

As a starting point of each refolding reaction, theind of solubilization strategy has to be considered.n case of chemical extraction at high denaturant con-entration refolding procedures can directly be carriedut. Starting from isolated inclusion bodies mostechniques aim to reach complete unfolding, which isest accomplished by chaotropic agents such as guani-inium chloride (GdnCl) or urea at high concentrationn combination with reducing agents. Depending onhe protein to be refolded higher final yield could bebtained by retaining certain native-like secondarytructure already present within the inclusion bodies.his could be achieved by using detergents (Purit al., 1992), buffers at high pH (Khan et al., 1998;ingh and Panda, 2005), GdnCl or arginine at lowoncentration (Tsumoto et al., 2003a) or even sodiumydroxide (Mahmoud et al., 1998; Suttnar et al., 1994)s solvent reagents. However this strategy is veryrotein dependent and has no general applicability. Assecond step refolding has to be initiated by removalf denaturant and providing conditions which allowntramolecular interaction and formation of correcttructure.

To ensure optimal yield proper refolding conditionsave to be found for every single protein. This is mostlyone in an empirical approach based on former experi-nce. If little is known about the protein of interest, thisay result in a vast number of different experiments

ince a large number of refolding additives have beenescribed in the past (De Bernardez Clark, 1998). Thisncludes denaturants in low concentration, polyols suchs sugars or sugar alcohols, ionic or non-ionic deter-

ents and organic solvents. They may either promoteolding of the protein or inhibit aggregation. The influ-nce of GdnCl and l-arginine has been investigated inetail by Umetsu et al. (2003). They have studied the

Dtaa

echnology 128 (2007) 587–596 591

olding behavior of antibody fragments at the presencef GdnCl, l-arginine and redox systems in a stepwiseialysis system. Different chemical and spectroscopi-al means were applied to determine aggregation, for-ation of structure, exposure of hydrophobic patches

nd formation of disulfide bonds. Effects of variousetergents and organic solvents on refolding yield ofysozyme were investigated by Yasuda et al. (1998)o find cheap refolding additives allowing processingt high protein concentration. Formation of aggregatesas monitored by dynamic light scattering. Again it has

o be emphasized that a positive effect of an additive onefolding of a certain protein may cause the oppositeor another one since protein properties are extremelyiverse.

In small scale refolding chaperones (Buchner et al.,992), folding helper proteins, artificial chaperonesMachida et al., 2000; Rozema and Gellman, 1996) andedox pairs such as GSH-GSSG are used to improveield. The use especially of chaperones is not practi-al in large scale, since they have to be available instoichiometric proportion to ensure efficacy. How-

ver, immobilization of folding aids allows a morefficient use of mostly expensive enzymes as shownith minichaperones (Altamirano et al., 1997), GroEL

nd GroES (Preston et al., 1999), oxidoreductases shuf-ing disulfide formation (Tsumoto et al., 2003b) orven a combination of chaperones (Altamirano et al.,999). Reuse of artificial chaperones has been reportedy Mannen et al. (2001). They used immobilizedyclodextrin to remove detergent from a denatured pro-ein to allow refolding. Operation of the process inirculating expanded bed mode allowed the strippingf detergent in a refolding requirement comparable tobatch suspension system but had the advantage of

calability.A fractional factorial design of the experimental set-

p significantly reduces the effort (Tobbell et al., 2002).dditionally automation of screening allows a first

valuation of different refolding conditions. Vincentellit al. (2004) describe an automated screening systemased on the detection of precipitation of proteins byurbidity measurements in a 96 well plate format. Theyssociate solubility of the protein to native structure.

ata provide valuable clues in terms of additive selec-

ion, however care has to be taken concerning solubleggregates or stable misfolded species, therefore anctivity assay is essential to draw further conclusions.

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92 A. Jungbauer, W. Kaar / Journal

n adjacent optimization of the refolding buffer isndispensable. This includes evaluation of minimumdditive concentrations providing a satisfactory yields well as possible exchange of expensive buffer com-onents to cheaper ones which is of specific concern forarge scale applications. Additionally temperature, pHnd of course redox potential may be crucial in a refold-ng reaction. Once proper conditions are found, optimaluffer exchange as well as protein concentration haveo be evaluated.

. Refolding by dilution

The simplest way to initiate refolding is to dilutehe unfolded protein in refolding buffer. Both the con-entrations of chaotrops and proteins are decreased insingle step therefore permitting intramolecular but

reventing intermolecular interaction. A final proteinoncentration of 10–100 �g/ml is generally applied inast dilution procedures. It is the method of choice inndustry because of simplicity of the process. Only atirred tank and feeding pumps are required. In the sim-lest setup only temperature has to be controlled. Theesolubilized protein from inclusion bodies is dispersedn the refolding buffer and the solution is kept for axed time, then the refolded protein is harvested. In

arge scale this technique poses several disadvantagesespite of the simplicity of the processing scheme. Forcale up of a stirred tank reactor mixing time shoulde kept constant. However this would be accompaniedith an extreme increase in power input, most time,ower per volume is kept constant, therefore mixingime increases with scale. Industrial scale devices have

ixing times lasting several minutes (Doran, 1995).niform and fast mixing as required for the rapidispersion of the feed stream is therefore hardly achiev-ble with common mixing devices and formation ofggregates may evolve from local high protein con-entration due to the imperfect mixing. An advancedixing reactor, the oscillatory flow reactor, was suc-

essfully applied for the refolding of lysozyme (Lee etl., 2002, 2001). In this device mixing intensity is cor-elated to an oscillatory Reynolds number ReO, which

s defined by reactor geometry, oscillatory frequencynd amplitude,

eO = DωxO

ν(5)

6

r

echnology 128 (2007) 587–596

here D is tube diameter,ω the angular frequency of thescillator drive, xO the oscillatory amplitude and ν is theinematic viscosity. The better scalability of the reactorn terms of mixing uniformity poses an advantage to atirred tank reactor.

Rendering dilution a continuous process in a com-arably small mixing device followed by a plug floweactor (Terashima et al., 1996) might pose anothernswer to improved scalability and offers the possi-ility of a direct connection to capture systems suchs expanded bed chromatography (Ferre et al., 2005).dditionally a continuous process allows the recyclingf aggregated protein and therefore an increase of yield.owever selection of operating parameters has to beptimized to retain productivity of a process as showny Schlegl et al. (2005a,b). Using a continuous stirredank reactor for refolding ultrafiltration devices haveo be introduced for the removal of chaotrops andeducing agents evolving from the solubilization ofnclusion bodies to retain a constant refolding environ-

ent (Hohenblum et al., 2004; Schlegl et al., 2005a,b).A consequence of low protein concentration are

arge volumes to handle. Expensive refolding bufferupplements as mentioned above have to be provided inigh quantity and wastewater treatment is another costriving factor in the process. Concentration steps haveo be included in the production scheme. This is eitherone by ultrafiltration or accomplished in subsequenturification steps such as ion exchange chromatogra-hy.

Higher final protein concentration could be reachedy pulse renaturation (Rudolph and Fischer, 1990) ored batch dilution (Katoh et al., 1999). Applicability ofhese methods is based upon the stability of native pro-eins, which exhibit less or no tendency to aggregateompared to folding intermediates. In an optimizedime scheme denatured protein is added to the reac-ion device. Intervals of protein addition or feed flowate are dependent on protein folding kinetics. Addi-ion of protein is limited to the amount of denaturanthich still allows refolding and has no negative effectn already folded protein.

. Pressure treatment

An alternative strategy is to use pressurized tanks asefolding reactors which is especially valuable for pro-

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A. Jungbauer, W. Kaar / Journal

eins with a high tendency to aggregate during refoldingr even during purification. Hydrostatic pressures of50–200 MPa permit the folding reaction while disfa-oring aggregation or completely reverse aggregationGorovits and Horowitz, 1998). Aggregates evolvingrom agitation, chaotrop-induced aggregates and bac-erial inclusion bodies were subjected to high pressurereatment at non-denaturing GdnCl-concentration (St.ohn et al., 1999). Even at protein concentrations up to.7 mg/ml high recovery rates of native protein could beeached. Additionally pressure treatment of inclusionodies for a longer period of time led to significantevels of active protein. P22 tailspike protein coulde refolded from aggregates (Foguel et al., 1999).xtended studies of folding and aggregation at dif-

erent pressure conditions were carried out and it washown that native protein was formed out of aggregatesfter pressure treatment even of short periods (Lefebvret al., 2004). Therefore this could also be used as aecycling strategy in a continuous refolding process.

Ligand binding domains of nuclear receptors, pro-uced as bacterial inclusion bodies, could successfullye activated by means of pressure refolding without these of denaturants, proving that also proteins, whichre prone to aggregation even in their native state cane refolded by this method (Schoner et al., 2005).he method permits refolding reactions at higher con-entration therefore reduces processing volumes andiminishes the need of chaotrops significantly.

. Large scale chromatographic refolding

Many chromatographic refolding procedures haveeen described in the past as reviewed recentlyJungbauer et al., 2004). Size exclusion based as wells adsorption based chromatographic refolding pro-edures have the advantage of operating at higherrotein concentration compared to conventional dilu-ion. However applicability in a large scale process isften restricted. Especially size exclusion chromato-raphic refolding on a batch column has limitations dueo small sample sizes and long run times. To achieveroductivity of a process it is therefore inevitable

o run it continuously. Schlegl et al. have character-zed a continuous matrix assisted refolding process ofovine �-lactalbumin (Schlegl et al., 2003) as well asf a recombinant therapeutic protein (Schlegl et al.,

tM

echnology 128 (2007) 587–596 593

005a,b) using annular chromatography at differentperating conditions including recycling of aggregates.n case of the aggregation prone therapeutic protein sig-ificant increase in yield could be reached comparedo a dilution process. Simulated moving bed (SMB)hromatography using four size exclusion columns wasuccessfully applied for the renaturation of lysozymes a model compound (Park et al., 2005). The processas designed using lysozyme and denaturant partition

oefficients obtained from batch column experiments.imulation of the process was in good agreement tobtained experimental results. However applicabilityf the process for the refolding of crude denatured pro-ein solutions remains to be shown. Sample application,hich is critical for successful size exclusion chro-atography, might create problems when using crude

xtracts and especially the continuous regenerationf the chromatography matrix, which requires ratherarsh conditions for the removal not only of unspe-ific bound protein aggregates but also other residualost cell compounds, poses a challenge to maintain thetability of the process.

Expanded bed chromatography is suitable for deal-ng with crude samples. Capture of the target proteinan be accomplished directly from cell homogenatess shown by Cho et al. (2001). They used an ionxchange matrix to adsorb a recombinant fusionrotein, wash out cell debris and unbound componentsnd exchange the buffer to initiate refolding, all inxpanded bed mode. Stability of the bed could beaintained by restricting the applied sample volume

herefore diluting the high molar urea buffer, whichould have higher density than the used chromatog-

aphy resin. Ion exchange in fixed bed mode haseen described as a valuable tool for chromatographicefolding processes (Creighton, 1990; Li et al., 2002;tempfer et al., 1996). A continuous process for theefolding of �-lactalbumin was described by Macholdt al. (2005). Folded protein could be separated fromggregated protein, which was recycled thereforencreasing the final yield of the reaction.

. Analysis of folded proteins

Recombinant proteins used as therapeutics haveo be proven to fulfill the required quality criteria.

ost important is, besides high purity, the activity

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94 A. Jungbauer, W. Kaar / Journal

f the compound which is closely correlated to itsative structure. Activity assays combined with circu-ar dichroism spectroscopy are classified as adequate

eans to prove the identity of the product. Typically,D spectra of unfolded, native and refolded proteinre compared and the latter ones have to show equaleatures.

The correct formation of disulfide bonds is fre-uently determined by reversed phase chromatography.ntermediate states, wrong as well as correct forma-ion of disulfide bonds can effectively be separatednd monitoring the folding status during the process isherefore possible (Goldenberg and Creighton, 1984;

u et al., 1998).Absence of aggregated protein has to be proven after

urification of the refolded protein. This may be accom-lished by analytical size exclusion chromatographyith on-line stray light detection. The intensity of scat-

ered light increases with the size of a compound inolution, therefore aggregates create a large signal andven traces can be detected.

. Conclusions

Albeit refolding by dilution is still the preferredechnology for large scale refolding, a lot of alterna-ive strategies have been developed in the past. Mostromising seem matrix assisted refolding using simplehromatography sorbents and pressure driven refold-ng. Still, the goal of future developments is to find

ethods to increase the final protein concentrationnder which refolding is performed.

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