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1 Introduction

Proteins can be considered to be amino acid poly-mers that are selected by natural evolution and foldduring and after synthesis into a three-dimension-al form called the “native conformation”. In fact, thenative conformation of a protein is neither uniquenor static; it is actually represented as an ensembleof subtly different polymer conformations that co-exist in equilibrium under certain physicochemicalconditions [1, 2].The conformation of a protein de-termines its bioactivity by providing exposed bind-

ing or catalytic sites by which the protein interactswith other cell components to exert its function.Changes in the protein’s environment can causechanges in the conformation of proteins, therebyaffecting their function. Moreover, the mechanismof action of most drugs depends on binding to spe-cific proteins (receptors, enzymes) to change theconformation with multiple functional conse-quences [3].

Defects in protein folding characterize a num-ber of human genetic disorders. For example, mis-folded proteins can accumulate into plaque-likescales called “beta sheets” that are related to dis-eases such as Alzheimer’s, Parkinson’s, and sometransmissible spongiform encephalopathies [4].Other proteins, such as the tubulin dimers of mi-crotubules, spontaneously switch from one confor-mational state to another as part of their normalfunction [5].

Review

Tools to evaluate the conformation of protein products

Bruno Manta1,2, Gonzalo Obal3, Alejandro Ricciardi4, Otto Pritsch3,5 and Ana Denicola1*

1 Lab. Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Uruguay.2 Grupo Biología Redox de Trypanosomas, Institut Pasteur de Montevideo, Uruguay3 Unidad de Biofísica de Proteínas, Institut Pasteur de Montevideo, Uruguay4 Laboratorio de Control de Biofármacos, Institut Pasteur de Montevideo, Uruguay5 Departamento de Inmunobiología, Facultad de Medicina, Universidad de la República, Uruguay

Production of recombinant proteins is a process intensively used in the research laboratory. Inaddition, the main biotechnology market products are recombinant proteins and monoclonalantibodies. The biological (and clinical) properties of the protein product strongly depend on theconformation of the polypeptide. Therefore, assessment of the correct conformation of the pro-duced protein is crucial. There is no single method to assess every aspect of protein structure orfunction. Depending on the protein, the methods of choice vary. There are general methods to eva-luate not only mass and primary sequence of the protein, but also higher-order structure. This re-view outlines the principal techniques for determining the conformation of a protein from struc-tural (biophysical methods) to functional (in vitro binding assays) analyses.

Keywords: Circular dichroism · Isothermal titration calorimetry · Fluorescence · Protein folding · Surface plasmon resonance

Correspondence: Dr. Otto Pritsch, Institut Pasteur Montevideo, Mataojo 2020, 11400 Montevideo, UruguayE-mail: pritsch@pasteur.edu.uy

Abbreviations: ANS, 1,8-anilinonaphtalene sulfonate; AUC, analytical ultra-centrifugation; CD, circular dichroism; CE, capillary electrophoresis; DLS,dynamic light scattering; DSC, differential scanning calorimetry; EF, elec-trophoresis; FTIR, Fourier-transformed infrared spectroscopy; HMW, highmolecular weight; ITC, isothermal titration calorimetry; LS, light scattering;SEC, size-exclusion chromatography; SPR, surface plasmon resonance;TPM/MS, trypsin peptide mapping via mass spectrometry; WB, western blot

Received 14 February 2011Revised 31 March 2011Accepted 4 April 2011

* Additional corresponding author: Dr. Ana Denicola, Instituto QuímicaBiológica, Facultad de Ciencias, Iguá 4225, 11400 Montevideo, UruguayE-mail: denicola@fcien.edu.uy

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The production of recombinant proteinsthrough DNA technology is a methodology usedevery day in many research laboratories and its usein the pharmaceutical industry is growing. Prod-ucts launched in the biotechnology market broadlyfall into four categories: (i) therapeutic recombi-nant proteins (including hormones, growth factors,and blood-related factors); (ii) monoclonal anti-bodies (murine and humanized); (iii) therapeuticvaccines; (iv) and nucleic acids (DNA/RNA thera-pies). Of these categories, recombinant proteinsand monoclonal antibodies are the leading classeson the market [6].

Obtaining a high yield of a soluble protein in theproper conformation is the ultimate goal in recom-binant protein production based on DNA technol-ogy. In the past 20 years, different methods havebeen developed and improved to achieve this goal,but the enteric bacterium Escherichia coli is by farthe most extensively used organism for the inten-sive production of proteins both in research labo-ratories and in the pharmaceutical industry. Eventhough several eukaryotic expression systems havebeen developed (yeast, fungi, insect, mammalian,and plant cells), E. coli still offers several advan-tages, including very well characterized genetics,inexpensive cell cultures, rapid biomass accumula-tion, simple scaleup process (see [7], which ap-pears in this issue). It is, however, not uncommonthat overexpressed recombinant proteins fail toreach the correct conformation (folding) and un-dergo proteolytic degradation or associate witheach other to form insoluble aggregates of non-na-tive (or partially denatured) proteins, known as in-clusion bodies [8]. Even though solubilization of re-combinant proteins is possible and has been suc-cessfully used in research and in the biopharma-

ceutical industry (see [9], which appears in this is-sue), special considerations and strict quality con-trol of the refolded protein are required. Eukaryot-ic expression systems can overcome the limitationsof prokaryotic ones, but protein yields are usuallyless and costs are higher.

When producing a recombinant protein, the pri-mary aim is to finally obtain a unique band in gelelectrophoresis (SDS-PAGE, see Table 1) and con-firm that it has the correct molecular weight andamino acid sequence by tryptic peptide mappingfollowed by mass spectrometry analysis (TPM/MS,Table 1). However, this approach cannot distin-guish between a completely unfolded, inactive, anduseless protein from one in the native, active con-formation1.Therefore, the conformation of the finalprotein obtained must be checked, especially if thefinal destination of the recombinant protein is foruse as a biopharmaceutical. In addition to produc-tion itself, the purification steps (lysis, precipita-tion, chromatography, concentration steps, etc.) canintroduce changes into the protein conformationthat can influence its biological (and clinical) prop-erties. We cannot disregard conditions of proteinproduct storage, handling, and distribution, that is,inappropriate buffer during storage or failure tomaintain the cold-chain that could alter the proteinconformation and result in inactive or, even worse,toxic products [10].

The polypeptide backbone can suffer chemicalchanges (e.g., cleavage of initial amino acid resi-dues, alternative disulfide bonds, methionine or

1 Although, under limited and controlled conditions, TPM/MS can givedifferent peptide profiles for proteins in different conformations.

Table 1. Structural characterization methods

Information Method / Techniquea)

Primary sequence Edman N-/C-terminal sequence TPM/MSSecondary structure far-CD FTIR NMR X-rayTertiary structure near-CD Fluo NMR X-rayQuaternary structure AUC SEC (D)LSSize (molecular mass) EF SEC MS AUCCharge IEC IEF CEHydrophobicity RP-HPLC HIC-HPLC FluoImmunoreactivity WB ELISA SPRGlycosylation CE MS (LC-MS/MS)

a) Abbreviations: AUC, analytical ultracentrifugation; CD, circular dichroism (far-UV region: 190–240 nm and near-UV region: 240–350 nm); CE, capillary electrophore-sis; (D)LS, static or dynamic (D) light scattering; Edman; Edman degradation, EF, electrophoresis in polyacrylamide gels (usually named as SDS-PAGE); ELISA, en-zyme-linked immunosorbent assay; Fluo, intrinsic (Trp, Tyr) and extrinsic (probes) fluorescence spectroscopy; FTIR, Fourier-Transformed Infrared spectroscopy;IEC, ion-exchange chromatography; IEF, isoelectric focusing chromatography; LC-MS/MS, liquid chromatography coupled to on-line MS/MS detection. NMR, nu-clear magnetic resonance spectroscopy (several applications); RP- and HIC-HPLC, reverse-phase (RP) and hydrophobic interaction (HIC) high-performance liquidchromatography; SEC, size-exclusion chromatography; TPM/MS, trypsin peptide mapping via mass spectrometry (MS); WB, western-blot; X-ray, protein crystallo-genesis and crystallography.

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cysteine oxidation, cross-linking, deamidation ofaspartate, cyclization of a glutamine chain, or mod-ification in the N- or C-terminal ends), so can thecarbohydrate moiety (if it is a glycoprotein) or theprosthetic group.

Membrane proteins are particularly importantbecause they represent the majority of biopharma-ceutical protein targets (receptors, ion channels,transporters) [3]. They are also the most challeng-ing for structural biologists, with less than 40 en-tries in the Protein Data Bank for eukaryotic mem-brane proteins [13–16]. In contrast to water-solubleproteins, their surface is relatively hydrophobicand they can only be extracted from the cell mem-brane with detergents. They are also often flexibleand unstable. This leads to challenges at all levels,including expression, solubilization, purification,and structure characterization. Recent advanceshave been made in the solubilization of membraneproteins by using a wide range of detergents andlipid–detergents mixtures that are then applied tomost of the biophysical methods developed for sol-uble proteins to assess conformation [14–16].

The need for regulatory protocols to assure thequality of the biopharmaceutical protein product isrecognized and some guidelines have already beendeveloped by the International Committee for Har-monization (ICH) of the US Food and Drug Admin-istration (FDA; guidelines Q6B, Q5E) and the Euro-pean Medicines Agency (EMA; Comparability ofmedicinal products containing biotechnology-derived proteins).

2 Functional versus structuralcharacterization

The best method to assess the right conformationof a protein product is to test its biological activityby using a specific bioassay for the protein tested.Bioassays, including cellular or animal models, testthe effect of the protein product in a biological sys-tem. However, bioassays are, in general, expensive,complex, and not always available in a research orbiotechnology laboratory (see below).An approachto study the functional activity of the protein wouldbe to characterize its enzymatic activity [11], if any,

or its interaction with a specific ligand, which isusually the first step in protein-induced cellular re-sponse. On the other hand, there is a battery of bio-physical methods, some of them simple and inex-pensive that are highly useful for testing confor-mation of every protein product (Table 1). Howev-er, there is no a single analytical method able toassess every aspect of protein conformation orfunction. In this sense, it is appropriate, and evendesirable, to apply more than one analytical proce-dure to evaluate the same quality attribute (e.g.,molecular weight, oligomeric state, secondary/ter-tiary structures). Each method should employ dif-ferent physicochemical or biological principles tocollect data for the same parameter and maximizethe possibility of detecting conformational differ-ences in the product.

We should note that it is not easy to fully char-acterize a protein product and most of the time it isnot necessary. But some criteria beyond SDS-PAGEand MS should be adopted to check the final pro-tein product, mainly because small changes inamino acid sequence due to mutations or even mi-nor modifications in protein production can resultin subtle changes in protein structure or conforma-tion that could compromise its biological activity[12]. Table 1 summarizes general physicochemicalmethods to evaluate different levels of proteinstructure and conformation.Table 2 groups some invitro biological methods that are more specific fortesting the functionality of the protein product.Some of these methodologies will be briefly de-scribed in the next sections.

3 Spectroscopic methods to evaluatesecondary and tertiary structure

Once a soluble recombinant protein is obtained, itis easy to run a UV/Vis spectrum. The signal in thearomatic region (around 280 nm) is not very in-formative about protein conformation unless thesecond-fourth-derivative of the spectra is analyzed(as examples, see [17–19]). In contrast, if the pro-tein does not have a prosthetic group, the near-UVregion (300–340 nm) can give an indication of a po-tential structural alteration leading to aggregation

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Table 2. Functional characterization methods

Information Methoda)

Enzyme activity Enzymatic assayBinding to specific ligand(s) SPR BLI ITC AC ELISA

a) Abbreviations: SPR, surface plasmon resonance; BLI, biolayer interferometry; ITC, isothermal titration calorimetry; AC, affinity chromatography; ELISA, enzyme-linked immunosorbent assay.

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that results in characteristic light scattering in thisregion relative to the buffer. If the protein has aprosthetic group that absorbs in the visible region,its spectral characteristic can also give informationabout folding, as exemplified for the recombinanthorse cytochrome c [20].

Proteins that possess aromatic amino acidresidues can be probed using intrinsic fluorescence(e.g., tryptophan). Fluorescence emission spectra(λexc = 280 or 295 nm) is easy to perform and a shiftin the wavelength maximum of emission (properly,center of mass of the spectra) is indicative ofchanges in the protein environment of the trypto-phan residues, thus, intrinsic fluorescence is verysensitive to changes in tertiary structure due to im-proper folding or denaturation [21]. For example,Sahu et al [22] produced His-tagged human phos-pholipid scramblase 1 in E. coli and purified it fromthe inclusion bodies under denaturing conditionsby using 8 M urea. The refolding of the enzymewhen urea was removed was detected by a 10-foldincrease in its intrinsic fluorescence and an 8 nmblueshift in the emission maximum.

The same conformational change can be detect-ed by quenching the tryptophan fluorescence withacrylamide or iodide (both hydrophilic quenchers;the former neutral, the latter negatively charged).Stern–Volmer constants can be calculated by fol-lowing the decrease in fluorescence intensity at in-creasing concentrations of the hydrophilic colli-sional quencher; this indicates the accessibility ofthe tryptophan residues to the solvent (as exam-ples, see recent reports [17, 22]).

Another valuable fluorescence technique is toevaluate protein superficial hydrophobicity by us-ing the fluorescent probe 1-anilinonaphtalene-8-sulfonate (ANS) (λexc = 370 nm, λem= 480 nm). Flu-orescence of ANS is very sensitive to the polarity ofthe environment, so its emission in an aqueous sol-vent is almost zero and dramatically increaseswhen the probe binds to hydrophobic binding sitesof the protein [23]. In recent work [24], heat-shockprotein 70 from yeast (Ssa1) was overexpressedin Pichia pastoris and conformation-dependenthydrophobicity was analyzed by ANS binding (ex-trinsic fluorescence) and complemented with in-trinsic fluorescence acrylamide quenching studies.Roman et al [15] recently studied the unfoldingequilibrium of a membrane protein (a coppertransporter from Archaeoglobulus fulgidus) usingfluorescence spectroscopy. The purified proteinreconstituted in phospholipid/detergent mixedmicelles in the presence of guanidinium hydro-chloride exhibited a decrease in fluorescence in-tensity; the center of spectral mass shifted from 342to 350 nm, The fraction of Trp fluorescence acces-

sible to the soluble quenchers shifted from 0.52 inthe native state to 0.96 in the unfolded state. Also,hydrophobic patches mainly located in the trans-membrane region were disrupted, as indicated byANS fluorescence [15].

Circular dichroism (CD, for a review, see [25, 26])uses incident circularly polarized light and is sen-sitive to chirality and asymmetry of the samplemolecule. Far-UV CD (190–240 nm) examines thepeptide backbone and can be used to estimate thefraction of secondary structure, whereas near-UVCD (240–350 nm) can characterize the environmentaround aromatic residues and disulfides and isparticularly sensitive for detecting changes in ter-tiary protein structure. In recent work [17], thestructure of different β-lactamase variants wereanalyzed and compared with the wild type by usingfar- and near-CD spectroscopy. Whereas nochanges in secondary structure were observed (al-most identical far-CD spectra), the near-UV CDclearly identified significant changes in tertiarystructure that were also confirmed by intrinsic flu-orescence (with blueshifts >30 nm) and acrylamidequenching [17]. The secondary structure of mem-brane proteins can also be assessed by far-CDspectroscopy if properly solubilized in micelles(see [15, 16] as examples).

Fourier-transformed infrared (FTIR) spec-troscopy is a versatile spectroscopic technique toanalyze secondary structure of proteins by follow-ing vibrations of the peptide group: amide I band(carbonyl stretching) at around 1650 cm–1 andamide II band (mainly N–H bending) close to 1540 cm–1 [27, 28].The vibration energy for amide Iand amide II bands will differ, depending on thesecondary structure of the polypeptide becauseα helix, β sheets, or turns will present differenthydrogen bonding that slightly alters the vibrationfrequency. Software is available to deconvolute theIR absorption band of the protein and analyze frac-tions of secondary structures, as reported previ-ously [24] for a study on conformational changes inrecombinant Hsp70 after nucleotide binding. It isinteresting to note that FTIR spectroscopy can notonly be used for soluble, but also for insoluble pro-teins, as in the case of partially denatured proteinsin inclusion bodies [29].

Finally, multispectroscopic monitoring can beperformed (far- and near-UV CD, FTIR, fluores-cence, and UV spectroscopy), coupled with detailedanalysis of spectra, that give more complete char-acterization of different protein conformations insolution [30].

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4 Tools to evaluate quaternary structure:Detection of aggregates or degradationproducts

Size-exclusion chromatography (SEC, also knownas gel-filtration, gel-permeation, or molecular-sieving chromatography) is generally used to char-acterize the size and distribution of species withinthe final protein product (for comprehensive re-views see [31, 32]). In addition, SEC can be used tostudy unfolded or partly folded conformationalstates of a protein [33, 34]. The eluate from a SECcolumn can be analyzed with online UV/Vis, lightscattering, or refractive-index detectors to give ac-curate molecular weight and species distributionsof the protein product (reviewed in [35]). It is par-ticularly useful for evaluating the oligomeric statusof the protein product, in addition to the potentialformation of either aggregates or degradationproducts [36].

Analytical ultracentrifugation (AUC; reviewedin [37, 38]) is a hydrodynamic methodology to ana-lyze biopolymers with respect to conformations insolution, conformational changes, association be-havior, and homologous or heterologous interac-tions that can also be extended to include the ther-modynamic characterization of protein–protein in-teractions. There has been a resurgence of AUC inbiophysics, mainly due to the upgrade of commer-cial analytical ultracentrifuges and the improve-ment of available software and algorithms to ana-lyze the experimental output. AUC can be used intwo ways: in sedimentation velocity the movementof the protein through a proper media at high cen-trifugal fields can provide information on size,shape, and interactions of the protein with itself orothers; whereas in sedimentation equilibrium, theprotein concentration gradients formed duringcentrifugation are analyzed to extract informationon molecular mass, stoichiometry/thermodynamicsof the association. It is extremely useful to study theself-association properties of proteins at equilibri-um [39] and how they can be modified by ligands ordrugs, as reported recently for tubulin [40].

Light scattering (LS) is a technique that can pro-vide the absolute molecular mass and size of nativeand non-native conformations of macromoleculesin solution. Briefly, LS techniques use a mono-/multiwavelength radiation source to irradiate aprotein sample in solution and measure someproperty (intensity, fluctuation, angles) of the lightscattered by the protein particles or aggregates (fora detailed description, see [41]).As noted previous-ly, LS is usually coupled to exclusion chromatogra-phy to determine the absolute molecular mass ofprotein [35].

Dynamic light scattering (DLS; also known asphoton correlation spectroscopy or quasi-elasticlight scattering) measures fluctuations in the in-tensity of light scattered from particles undergoingBrownian motion in solution and relates this toparticle size. DLS is considered to be a comple-mentary technique to SEC; while SEC is invaluablefor detecting low-molecular-weight (LMW)oligomers, the strength of DLS lies in its ability todetect very small amounts of high-molecular-weight (HMW) aggregates (as a example, see thework reported in [42] on heat aggregation of in-sulin).

Proteins can form aggregates in solution, most-ly due to oppositely charged subunits or hydropho-bic regions binding to form highly stable aggre-gates (see review [43], which appears in this issue).Since aggregation is a process governed by ther-modynamic and kinetics, it is a typical byproduct oflong-term storage of proteins and other biophar-maceuticals [44]. Aggregation can interfere withthe biological activity of a protein and develop aninappropriate immune response, which can bedangerous to the patient [12]. The aggregation ofproteins in solution can be properly measured withDLS because it is particularly sensitive to the for-mation of small amounts of protein aggregates. Forquality control of interferon (IFN) α-2 [45], the DLSresults for the IFN-α2a samples revealed that themajor species was the monomer (0–10 nm), withsome HMW aggregates in the 100–1000 nm sizerange. After normalizing by the volume of the scat-tering particle, the contribution from the HMWspecies was almost negligible (0.1–0.2%).

5 Stress stability studies: Thermal,denaturant, oxidation, and proteolysis

Another way to estimate the global conformation ofa protein is to study its susceptibility to the actionof denaturant or degrading agents, such as temper-ature, urea, oxidants, or several proteases. If theconformation of the protein is conserved, weshould expect the same response (unfolding, oxi-dation, proteolysis, etc) profile for each batch of theprotein tested. These assays (see Table 3) are ex-tremely useful in stability studies of protein prod-ucts upon storage, distribution, or aging.

The thermodynamic stability of a protein can beanalyzed by measuring the free energy difference(ΔG) between the folded and the unfolded states.The energetic contributions that keep a proteinfolded (hydrophobic packing, hydrogen bonding,and electrostatic interactions) are nearly offset bythe entropic penalization of the folding state, indi-

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cating that natural proteins are only marginallystable [46]. The current view is that proteins mustbe marginally stable to function; therefore, margin-al stability represents the results of positive selec-tion [47]. Due to this subtle balance between vari-ous physical interactions, minor modifications ofthe protein (oxidations, disulfides, partial proteoly-sis), or the environment (pH, temperature, ionicstrength), the equilibrium may shift between fold-ed and unfolded species with profound and some-times unpredictable effects on protein function[48].

Experimentally, ΔG values can be obtained fromdenaturing experiments in which the protein un-folds by increasing the temperature or the concen-tration of denaturing agents, such as urea andguanidinium chloride. CD is particularly suitable tomonitor thermal unfolding, especially for α-helicalproteins, because unfolding determines the disap-pearance of the characteristic negative ellipticityclose to 220 nm (for examples, see [33, 49]). Formeasuring thermal stability, CD requires less pro-tein than other methods, such as differential scan-ning calorimetry (DSC; see below). Intrinsic pro-tein fluorescence is generally used to follow chem-ical denaturation curves [21] and the concentrationof denaturant that determines 50% unfolding (Cm)is a reliable parameter to compare lot-to-lot stabil-ity [50].

An approach widely used is the addition of a flu-orescent probe that binds to the hydrophobic sitesexposed during unfolding. The fluorescence prop-erties of the probe are sensitive to the polarity ofthe environment (e.g., ANS, as described above; foran example, see [19]).

DSC can measure differences between heat ca-pacities of two solutions with continuous scanningover a defined temperature range. These solutionsare placed in the cells of the DSC instrument andare then heated or cooled at a fixed rate.The ener-gy required to maintain equal temperatures in bothcells is recorded as a function of temperature. Theconformation of compact, small, globular proteinsusually does not suffer major changes upon heat-ing to some critical temperature, but then unfold-

ing with extensive heat absorption can be meas-ured as increased heat capacity. In a single DSC ex-periment it is possible to determine Tm, the mid-point of the transition temperature where 50% ofthe protein is unfolded, the enthalpy (ΔH) of un-folding (integrating the area under the heat ab-sorption peak), and the heat capacity change (ΔCp)associated with unfolding [51].We should note thatif unfolding is not a reversible process (which, ingeneral, is the case), the only comparable data thatcan be extracted from unfolding curves is Tm (orCm) by taking the precaution of using the same scanrate [52]. In recent work, Niesen et al [53] reportedthat DSC may have the potential to estimate sam-ple quality for small, globular proteins of knownsize. By measuring 204 samples from 80 differentproteins, a positive correlation between unfoldingenthalpy and the size of small, globular proteinscould be used to estimate the quality of proteinpreparations.

Susceptibility to oxidation of a protein dependson its conformation and particularly to the relativeexposure of residues more sensitive to oxidation(cysteine, methionine, histidine). For example,Levine et al. [54] studied the susceptibility of re-combinant SHa prion protein to H2O2 treatment.Oxidized methionine residues were identified byMS after cyanogen bromide digestion (whichcleaves after methionine, but not after methioninesulfoxide). At its native conformation, four out ofthe nine methionine residues were easily oxidized.

Last, but not least important, is limited proteol-ysis. Limited proteolysis has been used routinely toassess the conformation of native or partly foldedstates [33] and, logically, can be used to comparelot-to-lot of recombinant protein production aswell as product stability. Interestingly, Heiring andMuller developed the ‘folding screen assayed byproteolysis’ method to exploit this type of analysisin high-throughput screening (HTS) [55].

Table 3. Stress stability studies

Information Methoda)

Thermal/denaturant stability UV CD Fluo DSCOxidation susceptibility Cys oxidationb) Met oxidationb)

Proteolysis susceptibility Trypsin and TPMc) Papain digestionc)

a) See the legend of Table 1 for definitions of the abbreviations.b) Cys or Met oxidation can be assessed by MS or other techniques.c) The proteolytic pattern of a protein can be studied with different proteases, depending on its primary sequence, post-translational modifications, and the informa-

tion required. Here, we note only two widely used proteases: trypsin (any protein) and papain (for mAb).

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6 Functional characterization: Ligand-binding interactions

One of the best ways to monitor the conformationof a protein product is to analyze its binding prop-erties. For example, antibodies bind to their anti-gens, and cytokines, growth factors, and hormonesbind to their cellular receptors.

While physicochemical techniques are veryvaluable in protein-conformation analysis, they donot address if the protein is functional or not. Thisis the reason why bioassays are an essential part ofthe biological activity characterization of any bio-therapeutic molecule [56].

Bioassays can be defined as analytical proce-dures for measuring the biological activity of a sub-stance based on a specific, functional, biological re-sponse of a test system [57]. In vivo bioassays in-volve the use of animal models, which is the onlysuitable methodology to characterize bioavailabili-ty, clearance mechanisms, catabolism, efficacy, andother pharmacological activities (pharmacokinet-ics and pharmacodynamics) that in vitro tests can-not provide. In vitro bioassays involve the use ofliving cells in which the product interacts with cel-lular receptors that trigger a signal that has a meas-urable endpoint. There is a continuum cellular re-sponse, from receptor binding to induction of cel-lular activity, that must be understood if a bioassayis to be relevant to the product.

Binding assays can be used as alternative ap-proaches to assess protein conformation throughthe characterization of protein–ligand interactions.Different formats of binding assays were designedto provide information on the fundamental proper-ties of this interaction (stoichiometry, kineticsand/or thermodynamics) [58, 59].

Various immunoassay formats can be used toanalyze protein–ligand interactions, includingELISA, radioimmunoprecipitation (RIP), and WB.Each assay needs to be appropriately designed andvalidated to produce meaningful results. It has tobe mentioned that antigen or receptor proteinscoated onto a plastic surface are usually unable toreproduce the binding conditions of a cell surface.In addition, immunoassays provide little informa-tion on the kinetics of the binding activity.

ITC provides a direct way to perform a completethermodynamic characterization of protein inter-actions and it has been established as the gold-standard method for directly measuring ligand-binding affinity and thermodynamics of an interac-tion [60]. Interaction between molecules eithergenerates (exothermic reaction) or absorbs (en-dothermic reaction) small amounts of heat.The de-velopment of new ultrasensitive ITC instruments

allows the detection of these small changes in heatand allows the thermodynamic characterization ofmolecular interactions with as little as a fewnanomoles of material [61]. These instruments in-clude an automated configuration that provides asample throughput of at least 50 samples per daywith a capacity to process as many as 384 samples.Thus, it is a useful method at initial stages of thedrug discovery and development process.

Each ITC experiment provides a complete ther-modynamic profile of the interaction, including thebinding constant (KA); the number of binding sites(n); and the variation of enthalpy (ΔH), entropy(ΔS), and free energy (ΔG). Since heat is universal-ly generated or absorbed during any molecular in-teraction, ITC may be viewed as a universal detec-tor for such interactions. ITC may be used to studyinteractions by using native, modified, or immobi-lized substances and is the only bioanalyticalmethod that directly measures enthalpy.

By taking into account these considerations, ITCcan also be considered as part of a toolbox of bio-physical techniques useful for assessing the quali-ty and reproducibility of protein preparations byevaluating protein functionality [62–64].

Surface plasmon resonance (SPR) biosensorbinding assays are based on the measurement ofchanges in SPR, detected as changes in reflectedpolarized light following the binding of analytes toligands embedded in a dextran matrix on the sur-face of a gold-plated sensor chip [65, 66]. SPR oc-curs in a thin metal film under conditions of totalinternal reflectance and can be measured as a de-crease in light intensity at a specific angle. Whenmolecules bind to immobilized ligands on the sur-face of a gold-plated sensor chip, a shift of the inci-dent light angle at which resonance occurs can bedetected by light sensors. By using this technology,we can obtain full kinetic parameters that charac-terize an interaction, such as association (kon) anddissociation (koff) kinetic constants.

The binding properties of many biotechnologyproducts have been characterized by using thistechnology and parameters for product analysishave been defined and validated [67–70].Thus, SPRmeasurements can also be considered a usefultechnique to assess the quality of protein prepara-tions by evaluating binding functionality.

By using SPR technology, it is now possible todetect the binding of cells to immobilized ligands,representing a highly ‘natural’ binding format thatcan detect interactions between cell-membrane re-ceptors in their native conformational state [71].

SPR technology is also useful for the detectionof conformational changes within a protein ad-sorbed to the sensor surface. In this situation, there

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is no change in the total mass of protein adsorbedto the matrix. The changes in biosensor signals inresponse to the conformational transition are muchsmaller than those resulting from protein–ligandinteractions.These changes reflect variations in theoverall environment close to the sensor surface andthe effect is not restricted to direct consequences ofprotein isomerization [72]. For example, the workof Flatmark et al [73] shows that SPR can measurethe global conformational transition in humanphenylalanine hydroxylase in response to sub-strate binding and catalytic activation.

7 Conclusions and perspectives

Recombinant proteins are being intensively usedin many biotechnology research laboratories, aswell as in industry, of which biopharmaceuticalsleads the group. It is important to assess that prop-er folding of the final protein product is achieved.No single method is able to assess every aspect ofprotein structure or function. We presented anoverview of methods available to evaluate the con-formation of proteins.This is not exhaustive, but in-cludes the most accessible techniques, indicatingadvantages/disadvantages and the type of informa-tion they provide. Besides electrophoresis (SDS-PAGE), WB, and MS analysis of the protein band, aphysicochemical as well as a biological characteri-zation of the product is necessary. The methods ofchoice will strongly depend on the protein tested.

Table 4 gives a summary of various techniquesused to monitor protein conformational changes,along with some practical characteristics, such as

sample consumption, time of assay, and potentialhigh-throughput setup.

We have omitted from this review the two morepowerful techniques available to study the confor-mation of proteins: NMR spectroscopy and X-raycrystallography.These two approaches are unques-tionably leaders in the field of structural biology,but they still require very sophisticated equipment,appreciable amounts of sample, and specialist hu-man resources that keep them away from thepipeline in protein production and control.

Newer techniques (or new applications of well-established techniques) are being developed tocomplement these traditional biophysical assaysand to provide information on the success of pro-tein folding.

A novel use of MS was reported to follow proteinstability [74]. This method is based on hydrogen/deuterium exchange coupled to MALDI-TOF. Thechange in mass was followed with the time ofexchange at different concentrations of denaturingagent.

CE-based methods (reviewed in [75]) offerhighly efficient separation and analyses of biomol-ecules. When coupled to laser-induced fluores-cence detection (CE-LIF), the detection sensitivityis significantly improved [76].

BLI expands the capabilities of biosensors inthe detection of label-free interactions of biomole-cules in biological media without the need for aspecific system for handling microfluids [77] .

Herein, we focused on the most common meth-ods available to assess the conformation of a pro-tein product. Analytical methods give informationon different levels of structure of any polypeptide,

Table 4. Experimental considerations for choosing an analytical method

Methoda) Amount of sample neededb) Assay timec) High-throughputd)

AUC H h–dayCD M h

(D)LS M–H min–h +DSC H hEF L min–h +

Fluo L min–h +FTIR M min–hITC H h +MS L min–h +SEC M hSPR L h +

a) See the legend of Table 1 for definitions of the abbreviations.b) Amount of sample needed is arbitrarily classified as low (L), meaning 10 ng to a few μg of pure protein; medium (M), in the range 100 μg to 10 mg; and high (H),

meaning more than 10 mg of pure protein.c) Assay time is the minimum amount of time needed to perform an experiment and analyze the results, assuming that the experimental setup is already working and

that the operator is proficient in the technique. It is arbitrarily classified in three categories: from minutes to a few hours (min–h), several hours (h), and from hoursto days (h–day).

d) Some applications of the methods marked with “+” were already set for HTS.

© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 9

whereas in vitro binding assays are specific foreach protein tested, indicating its potential biolog-ical activity.As mentioned before, no single methodis sufficient to fully characterize the protein con-formation. A combination of the methods outlinedabove can provide information indicative of thesuccess of folding of the protein product, and im-portantly, allow in vitro comparison of lot-to-lot in-tegrity.

This work was supported by Comisión Sectorial deInvestigación Científica (CSIC), Universidad de laRepública, and Agencia Nacional de Investigación eInnovación (ANII), Uruguay.

The authors have declared no conflict of interest.

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Ana Denicola is Head of the Physical

Biochemistry laboratory at the Chemi-

cal Biology Institute of Faculty of Sci-

ence, University of the Republic (Ude-

laR), Uruguay. She graduated in Chem-

istry/Pharmaceutical Chemistry at the

UdelaR, Uruguay and received her

Ph.D. in Biochemistry at Virginia Tech,

VA, USA. She is now full Professor of

Physical Biochemistry at the Faculty of Science, UdelaR. Her major re-

search interests concern mechanisms of bioproduction and reactivity

of oxygen and nitrogen species, in particular, the structural and func-

tional characterization of oxidative modifications of proteins.

Otto Pritsch received his M.Sc. in Bio-

chemistry in 1992 from Facultad de

Ciencias, Universidad de la República,

Uruguay. In 1997, he received his Ph.D.

in Immunology from Université Paris

VII, in France. After doctoral and post-

doctoral studies at the Institut Pasteur

de Paris, he received, in 1998, a posi-

tion as Associate Professor at the

Biochemistry Department, Facultad de Medicina, Universidad de la

República, Uruguay. In 2006 he became Associate Professor of the

Immunobiology Department. Since 2006 he has been Head of the Pro-

tein Biophysics Unit at the Institut Pasteur de Montevideo, Uruguay.

At present, his research is focused on studying human and animal

B-cell leukemia and particularly on the analysis of retroviral proteins

involved in animal leukemia pathogenesis at the molecular and

structural levels.

© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 11

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