Journal of Immunological Methods 405 (2014) 154–166
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Journal of Immunological Methods
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Research paper
Effective protocol for the investigation of physicochemical andconformational stability and aggregation kineticsmeasurements of therapeutic IgG2 monoclonal antibody
Ali Aboel Dahab⁎, Dhia El-HagDepartment of Pharmacy, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK
Article history:Received 29 October 2013Received in revised form 29 December 2013Accepted 31 January 2014Available online 13 February 2014
Characterisation of monoclonal antibodies (mAbs) represents an ongoing challenge due totheir diverse 3-dimensional structures that can affect their stability, immunogenicity and/ortoxicity. Although circular dichroism (CD) spectroscopy provides rapid determinations ofprotein secondary structure in solutions, there is a pressing need for an improvement in currentpractices in applying the technique for batch QC. There is a lack of experimental evidence in theliterature which is concerned with improving the current practices. This work is based on aneffective protocol for the study of IgG2a stability in solution using the simultaneousmeasurementsof absorbance, turbidity and CD. A novel approach has been developed for the study of the effectsof pH and additives with minimum protein shock that may cause premature aggregation anddeceptive results. A computer programme has been designed for the rapid and simultaneousanalysis of aggregation during UV and CD measurements, also, highlighting instrumentalvariations. Temperature stability determination, elucidation of unfolding pathways andaggregation kinetics were estimated with accuracy. This experimental approach providesimportant information about mAbs physicochemical and conformational stability, helpsdistinguish between unfolded, refolded, aggregated, and flocculated states and is an excellenttool in the development of therapeutic antibodies.
Keywords:AggregationAbsorbanceTurbidity and circular dichroismmonoclonal antibodies (mAbs)IgG2aStability
1. Introduction
The in vivo use of biopharmaceuticals particularly mono-clonal antibodies (mAbs) has been the concern of pharma-ceutical companies for many years (Wang et al., 2007).Although the early studies focused primarily on cancer, thefield has branched out to include most major forms of humandisease. Even though some mAbs have demonstrated clinicalutility and are now commercial products such as infliximab,rituximab, trastuzumab, bevacizumab, adalimumab, cetuximab,and palivizumab (Reichert, 2009) or in advanced stages ofdevelopment, many others are still undergoing preclinical and
early clinical development and evaluation. Biopharmaceuticalsare produced in prokaryotic (no distinct nucleus) or eukaryotic(distinct nucleus) cell systems (Schellekens, 2002). The finalmaterial has to be formulated with complex mixtures of excip-ients to ensure stability, in addition to freeze drying the finalproduct to avoid loss of activity during storage (Ohtake et al.,2011). An important aspect ofmAbpreparations is their physico-chemical stability upon storage (Abdul-Fattah et al., 2007).Nevertheless, during manufacturing, storage, and administra-tion, mAbs are subjected to various environmental stressessuch as solution pH, temperature, shear, and freeze–thaw.All methods of these complicated production and purifica-tion processes can greatly influence the nature and thequality of the final product. Therefore, understanding andidentifying optimum conditions to stabilize mAbs is a criticalstep for their successful development as therapeutic drugs.
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Optimizing solution pH, ionic strength, and adding phar-maceutical excipients are among formulation variables avail-able to minimize protein degradation, they can influence therate, pathway and extent of antibody aggregate formation. Inaddition, the composition of the formulation can have a directimpact on antibody self-association and solution viscosity,particularly at high antibody concentrations (Yadav et al.,2010; He et al., 2011). A systematic approach for identifyingoptimized, stable formulation of an antibody typically involvesscreening a series of pH conditions and excipients (types,concentrations, and combinations) andusingmethods to detectboth physical and chemical changes.
To be able to guarantee the physical and chemical integrity ofcomplex proteins such as mAbs, the availability of relevantanalytical techniques is prerequisite. It has been demonstratedby several analytical techniques, that the stability of IgGmAbs, isclearly pH-dependent (Bolli et al., 2010). Protein aggregation canbe either reversible or irreversible and can be induced by low orhigh ionic strength, heating, extremes of pH, freezing andthawing, and mechanical forces (Hawe et al., 2012). Moreover,it is important to take into account that local conformationalstability and fluctuations of partially altered structures caninfluence the aggregation propensity of IgGs (Thakkar et al.,2013). On the other hand, a fact that complicates the investiga-tion of the acid induced precipitation of mAbs is the apparentirreversibility of the precipitation process. A proper analysis ofthe precipitates is hampered, because most techniques forstudying the physical and chemical integrity of proteins cannotbe applied to precipitated material. An approach to circumventthis problem is to study the events preceding precipitation.However, the simultaneous measurement of absorbance,circular dichroism and turbidity offer great advantages instudying various aspects of protein stability in solutionincluding elucidation of protein secondary/tertiary structurechanges and ease of routine measurement with relativelylow concentrations. Also, the CD measurement is less proneto being affected by the presence of the surrounding solutionand there are well-defined procedures available to estimateprotein secondary/tertiary structure content based on referencespectra of the different conformational elements (Greenfield,2006). The most widely used applications of protein CD are todeterminewhether an expressed, purified protein is folded, or ifa mutation and/or environmental changes (e.g. temperature,pH, additives) affect its conformation or stability, as well as thestudy of protein interactions.
The complete CD spectrum of a protein can be subdividedinto three regions based upon the need tomaintain absorbance(A) = 1.0 for good measurements. CD associated with aro-matic amino acid side chains and disulfide (\S\S\) is foundin the wavelength range 320–240 nm (near UV), using con-centration of the order 0.5 mg/ml in a 1.0 cm cell. This regionprovide information about the local environment of the relevantresidues. In the far UV (240–185 nm) using 0.2–0.5 mg/ml in a0.05 cm cell, the CD spectrum provides information about thepeptide back bone conformation (secondary structure). Ingeneral, the CD spectrum is sub-divided into 2 main regions(Fig. 1) and careful consideration is needed to match concentra-tion and pathlength to the wavelength being studied.
In this study, we have developed an effective protocol tostudy the integrity and stability of therapeutic mAbs insolution as a function of pH, excipients and temperature
employing the simultaneous measurement of CD, absorption,and related light scattering (turbidity). Subsequently, we per-formed spectroscopic and aggregation-based kinetic studies bymeasuring UV and CD variations and turbidity increases. Thesemeasurements were used to identify the optimum solution pH,temperature and excipients' concentration that inhibit turbid-ity and aggregate formation. Because of the lack of informationin literature which deal with practical considerations intitration procedures and based on experimental observations,a novel approach was developed and utilised for the pH andadditives titrations. On the other hand, the selection ofstabilizing conditions from considerations of aggregation/turbidity analysis under accelerated conditions is correlat-ed with the selection of stabilizing conditions basedon structural transition data obtained from UV and CDspectroscopy. A better understanding of the relationshipbetween spectroscopic variation kinetic measurementsand biophysical conformational stability data not onlyprovides insight into mAb physicochemical stability butalso enables identification of stabilizing conditions in arapid, high-throughput manner as part of a more efficientapproach to mAb formulation development.
2. Materials and methods
2.1. Materials
The IgG2a mAb solutions were supplied by Pfizer PLC(Sandwich, Kent, UK) at concentrations between 3–11.9 mg/mland pH 5.5. The protein was diluted to 0.5 mg/ml or otherindicated concentrations in distilled water obtained from KCL.Sodium chloride, hydrochloric acid and acetic acid were Analargrade from BDH laboratory suppliers, England, UK. Sodiumacetate trihydrate (CH3COONa·.3H2O) was Analar grade fromHopkin &Williams Ltd., England, UK. Polysorbate 80 wassupplied by Pfizer PLC. Sodium hydroxide was obtained fromKoch-Light Limited, Suffolk, England, UK. The cells used in thisstudy were specifically manufactured for circular dichroismmeasurements from Hellma GmbH & CO.KG, D-79-371,Mullheim, Germany.
2.2. Methods
2.2.1. UV and CD spectroscopyA Jasco 720 CD spectrometer from Jasco International
Co., Tokyo, Japan was used in the study of the antibody'sphysicochemical stability. Using the fundamentals of simul-taneous measurements of absorbance and CD as describedby Aboel Dahab et al. (2010), measurements were mademainly in two regions, near UV (360–240 nm) and far UV(240–190 nm) using 1.0 cm, 0.2 and 0.05 cm pathlengths.Preliminary experiments were performed to evaluate theoptimum experimental parameters. Subsequently, it was con-cluded that excellent low noise spectra could be obtained usinga spectral bandwidth (SBW) as high as 5.0 nmanddata step sizeas great as 1.0 nm. However, using these parameters can causedistortion, but for monitoring processes, great time saving canbe achieved. On the other hand, for good specification doc-uments, a SBW of 1.0 nm and data step size of 0.2 nm provide agood compromise. The instrumental parameters that were usedare summarized in Table 1. All spectra were corrected by
Amide backbone
Tryp & -S-S-
0.05 cm cell
0.2 cm cell
Phe, Tyr, Tryp
1 cm cell
1 cm cell
-S-S-
(Local conformation)(Secondary structure)
Side-chainBackbone conformation
Near UVFar UV
Wavelength (nm)
180 200 220 240 260 280 300 320
Fig. 1. Main regions of protein CD and the associated pathlengths.
156 A. Aboel Dahab, D. El-Hag / Journal of Immunological Methods 405 (2014) 154–166
subtracting signals from buffer blanks. Data processing andanalysis were performed using the Jasco standard analysissoftware and Origin6 software packages. Also, protein structurewas displayed using Swiss PDB viewer and POV-ray.
2.2.2. Measurement guidelinesIt is important to follow a defined protocol for the simul-
taneous measurements of CD and absorbance to ensure thatthe data are trustworthy. (i) At the beginning of a series ofmeasurements, the CD spectrum should be measured overthe required wavelength range with nothing in the cell com-partment and the CD and HV0 (I0) data should be saved toprovide reference for subsequent absorbance measurementsand a monitor of the instrument and lamp status. These dataalso provide reference data to assess the suitability of themeasurement cells filled with solvent. (ii) To measure mini-mum quantities without too much attenuation of the lightintensity, a 1-ml sample is accommodated in a 1 × 1 cm strain-free cell. A Hellma cell would seem to be a good compromise,however, it is important to ensure that suitable beam constric-tion (iris) is in place to avoid the solution meniscus being in thelight path. (iii) The absorbance profile of the solvent providescriteria for the transparency and quality of both the solvent andthe measurement cell. For a perfect cell and non-chiralsolvent, the solvent CD spectra should be identical with theCD measured in step (i). Solvent measurements whereAsolvent N 0.5 indicate that the solvent is probably absorbing
Table 1Optimum experimental parameters for mAb measurements.
Pathlength (cm) 1.0Scan range (nm) 360–230Step size (nm) 0.2Response time (sec) 4Spectra bandwidth (nm) 1Sensitivity (mdeg) 20-50Scan speed (nm/min) 10-20
too strongly for good CD measurements. (iv) Measurementsshould be rejected if the total solution absorbance(Asolution) N 1.5. (v) For turbidity measurements, the distancebetween the sample and the detector should be carefullyconsidered. (vi) The concentration/pathlength combinationsindicated in this study are guidelines. For greater penetrationinto the far-UV (b200 nm), shorter pathlengths (and lowerconcentrations) may be required.
2.2.3. Determination of IgG2a mAb optimum pHThe extreme pH of a titrant drop can be sufficient to
promote antibody (seed) aggregation. Also, the addition oflarge amounts of a weak acid or base to the mAb solution cancause analyte dilution. Therefore, to avoid acid/alkalineaggregation or unwanted dilution, three approaches weretaken towards mAb pH titration to deduce the best way forpH studies:
I Conventional pH titration of mAbs: This titration is bestachieved by taking a sample and progressively adding atitrant in an appropriate manner and mixing with minimalstirring to avoidmechanical aggregation. Unfortunately, theresults were deceptive, as there was visual evidence ofparticle formation throughout the titration, which distortedthe spectroscopy. The CDof the denatured antibodywas notconsistent with a “random” coil structure. However, theacid denaturation spectra showed an intense negative CDband at 217 nm indicating a new β-sheet formation. The
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plot of ΔA294nm values vs. pH exhibited more than one pKa
values at ~3.7 and ~6.5 indicating the different ionisationstates of the aromatic side chain residues.
II Gentle single pot pH titration: This method provides aminimum quantity, and a “gentle” pH titration procedure.The mAb is diluted in acidified water at ~pH 2.5 (orpH 10.0) and the pH is taken towards pH 12.0 (or pH 2.0)with a dilute acid/base (30 mM HCl or 30 mM NaOH).However, this method suffers from the inability tomonitor each individual pH with time.
III Gentle multi-pot pH titration: This method requires0.5 mg/ml antibodies for every pHmeasurement. Dilutealiquots of the original antibody solution in pHpre-adjustedwater or buffer solutions are used. It is the sameapproach asin II except for using a separate mAb solution for each pHmeasurement to avoid premature aggregation and monitorpH variation with time. A complete pH titration requiresabout 5.0 mg (0.5 mg/1 ml aliquots) of mAb which couldbe critical if only limited quantities of mAbs are available.However, this was not the case here and this methodproved to be the most suitable and accurate approach fordetermining mAb optimum pH avoiding shocking theprotein into premature aggregation and was used in thisstudy.
2.2.4. Excipients' effect on stability of IgG2a mAb at fixedtemperature
UV and CD measurements at the near and the far UVregions were used to determine the effect of polysorbate 80and the effect of NaCl concentration on the stability andaggregation of IgG2a mAb at room temperature and optimumpH.
2.2.4.1. Polysorbate 80 (PS 80). Polysorbates are nonionicsurfactants widely used in the development of proteinpharmaceuticals as stabilisers. Different concentrations ofPS80 (0.2 mg/ml–0.8 mg/ml) were used to study its effecton the antibody stability at a fixed pH (5.5). Aliquots of theantibody solution (0.5 mg/ml) were dissolved in pre-preparedacetate buffer solutions with the required concentration ofPS80. Depending on these results, the pH effect on IgG2a mAbwas investigated in the presence (0.2 mg/ml PS80) and theabsence of PS80.
2.2.4.2. Sodium chloride. The effect of NaCl on IgG2a mAbstability at fixed pH and temperature was investigated usingthe UV and CD spectra in the near and far UV regions. Themost appropriate NaCl titration of mAb was achieved bypreparing a series of solutions of NaCl in 20 mM acetatebuffer pH 5.5 at various concentrations (0.0 M–4.0 M) inwhich a dilution of 0.5 mg/ml of IgG2a mAb was prepared.This was achieved by preparing two solutions (A, B). SolutionA contains zero salt concentration and solution B containshigh salt concentration. Equal increments of solution B wereadded to solution A after withdrawal of the same amountfrom solution A to achieve the required salt concentrationwhile keeping the mAb concentration in solution A fixed. Thisaddition/withdrawal method was performed to a separatesolution for each salt concentration to accurately obtain morerepresentative results for the effect of NaCl on IgG2amAb andavoid protein shock, salting out and aggregation.
2.2.5. Thermal stability measurementsCD signals from 350 nm to 240 nm (1.0 cm cell) and from
240 nm to 200 nm (0.05 cm cell) for the near and the far UVregions respectively at various temperatures were collectedusing Jasco spectrometer, J-720. The temperature regulationwas carried out using a Hewlett-Packard GMBH (89090A)Peltier system (Germany), with an independent thermocou-ple in the cell monitored by a metre. Antibody stock solution(11.9 mg/ml) was diluted to 0.5 mg/ml in 20 mM acetatebuffer pH 5.5, PS 80 0.2 mg/ml and 140 mM NaCl. Thetemperature range used was from 20 to 85 °C and thespectra were collected for each temperature change. Themelting temperature (Tm) and the onset temperature ofthermal unfolding (T0) were determined by plotting atemperature curve, which is then analysed using non-linearanalysis based on the thermal equilibrium and Van't Hoff 'sisochore equation.
2.2.6. Light scattering (turbidity)At wavelengths where a sample does not absorb, the ab-
sorption should be zero. Protein aggregates, suspended insolution, can present distorted optical spectra due to lightscattering/turbidity. In circularly polarised light, light scat-tering will be differential quantities involving the differencebetween left and right circularly polarised light (Wallace andMao, 1984). Circular intensity differential scattering (CIDS) isa macroscopic property associated with larger molecularentities involving a chiral array of many chromophores, whichcan dominate the normal molecular CD. Light scattering at theanalytical wavelength can be estimated by extrapolation frommodelling at another wavelength range. A portion of thespectrum where the apparent absorbance is due only to lightscattering is selected. A polynomial is fitted to this part of thespectrum using a least square fit to the logarithm of theabsorbance: A = a λn and log (A) = log (a) + n log (λ).
Where A is absorbance; λ is wavelength; n is the order ofthe relationship between absorbance and wavelength and ais a constant. The background absorbance at all otherwavelengths can be estimated using the coefficients deter-mined from the fit. These values are then subtracted from themeasured values to give the absorbance due to the analyte, asshown in (Fig. 2).
A computer programme that calculates light scattering inUV measurements has been devised (Aboel Dahab, 2013); itenables a reliable and a fast way of light scattering correctionby only entering the value of λ where the molecule does notabsorb to produce the scattering spectrum which is thensubtracted from the measured absorbance spectrum. Themeasurement of turbidity (light scattering) is dependent onthe distance between the sample and the detector. Movingthe sample from a position far from the detector to a positionclose to the detector window will see an apparent reductionin Asc. Similar measurements in the CD mode may also causedistorted spectra. Turbidity measurements at 400 nm of IgG2amAb aggregation resulting from pH variation were performedsimultaneously during absorbance and CDmeasurements. Thismethod saved a great deal of time and reduced the amount ofsamples required for analysis. Aggregation (turbidity) wasmeasured at different distances from the detector; 22.0 cm(far), 15.0 cm (mid), and 5.0 cm (near) from the detector.
0.0
0.2
0.4
0.6
0.8
1.0
Measured spectrumExtrapolated scatter spectrum
Ac= A
t - A
Sc
Asc
At
Ac
λ
Selected wavelength rangeA
bsor
banc
e
Wavelength (nm)
200 250 300 350 400
Fig. 2. Graph showing light scattering correction (background modelling)where Ac denotes corrected absorbance, At is total measured absorbance andAsc denotes absorbance due to light scattering (turbidity).
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3. Results and discussion
3.1. Structure and interpretation of spectra of the intact IgG2amAb
The refined structure of the intactmouse IgG2amAb (Fig. 3)was taken from the Protein Data Bank and is displayed usingSwiss PDB viewer and POV-ray. The grey areas represent thedifferences in amino acid residues and the aqua areas representpossible deletions during the biological synthesis of the anti-body. Tryptophan (blue) and tyrosine (red) residues have alsobeen highlighted due to their spectroscopic importance inprotein monitoring and characterisation.
The UV and CD spectra of the IgG2a mAb in the near andfar UV regions are displayed in Fig. 4. In the far UV region, theantibody exhibits a negative CD at ~217 nm and a positive
Fig. 3. The refined structure of the intact IgG2a mAb taken from the RCSBProtein Data Bank (displayed using Swiss PDB viewer and POV-ray).
one at ~198 nm which is characteristic of β-sheet conforma-tion. It is clear that the β-sheet is themost dominant secondarystructure element present in this antibody, indicating that itbelongs to the all-β IgG sub-class. The negative CD feature at~230 nm reflects Tryp absorbance, which indicate the contri-bution of aromatic residues to the far UV spectra of this IgGmolecule (Ignatova and Gierasch, 2007). The absorbance andCD spectra of IgG2a mAb in the near UV region from 250 to300 nmcan be used to elucidate the tertiary structure, quantifythe aromatic content of proteins and to monitor subtlestructural changes providing information regarding theexposure of the aromatic residues to the solvent. Aromaticresidues (Tryp, Tyr and Phe) also contribute to the far-UVspectra of a protein (Zhang et al., 2009), however, ingeneral, the contribution is very small but when the contentof these residues is very high the estimation of the secondarystructure becomes complicated.
Themost common transitions in protein absorption spectraare nπ* and ππ*. The two primary absorption bands for Trypare ππ* and occur at ~280 and 292 nm. The Tyr absorbancemaximum due to the ππ* transition is observed at ~275 nm.Finally, the UV absorption spectrum of Phe is often character-ized as weak and may be obscured by Tyr, however, significantstructure is usually observed and multiple bands can beresolved in the 240–270 nm region. In Fig. 4, the shoulder inthe absorbance spectrum at ~292 nm represent Tryp residue,which corresponds to the small positive CD peak at the samewavelength. Aromatic residues can exhibit circular dichroismwith ππ* absorption at ~250–300 nm. The Tryp environmentis reflected at 290–300 nm, and the Tyr environment at275–282 nm where the fine structure at longer wavelengthsmay be obscured by that from Tryp (Sreerama et al., 1999). Phehas signals as a triplet at 258, 264, and 270 nmwhich representdifferent vibrational levels of the excited state (Cleland et al.,2001). In Fig. 4, the fine structure of Phe CD, althoughweak, it isapparent at the previously stated wavelengths and the weakCD band at ~282 nm is more likely representative of Tyr. Theintensity and shape of the CD spectra can be influenced by therigidity of the protein, the nature of the environment in termsof hydrogen bonding, polar groups and polarisability, also,the more highly mobile side chains have lower intensities.Disulfide bonds also have circular dichroism related to nσ*transition at approximately 260 nm. Generally, the peak fora disulfide bond is wider than that for an aromatic residueand is usually quite weak. However, the intensity dependson a number of factors including the dihedral angle of thedisulphide bond (the C\S\S bond angle) and the effects ofneighbouring groups. These factors also affect the position ofthe absorption and CD bands.
3.2. Biophysical measurements and most stabilising conditions
Solution pH is an important variable among formulationvariables used to minimise protein degradation. A systematicapproach for identifying optimized and stable formulation ofan antibody typically involves screening a series of pHconditions. As mentioned earlier in Section 2.2, a gentlemulti-pot pH titration was performed. Fig. 5 shows the UVand CD spectra of IgG2a mAb as a function of pH in thepresence of PS 80. The magnitude and the extent of pHvariation depend on the protein structure and the relative
Fig. 4. UV and CD spectra of 0.5 mg/ml IgG2a mAb in 20 mM acetate buffer pH 5.5 containing 0.2 mg/ml PS80 and 140 mM NaCl.
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proportion of the amino acids. The near UV spectra show nosign of turbidity and there is no significant change in the pHrange 4.10–8.0. This indicates that in the presence of PS 80,the IgG mAb is fairly stable in this pH range. Above pH 8.0,
0.0
0.5
1.0
1.5
-0.0002
0.0000
0.0002
0.0004
pH 2.90
Abs
orba
nce
pH 2.90
pH 4.10
pH 11.50
pH 5.80
p
ΔA
Wavele
pH 9.0
200 220 240 260
200 220 240 260
Fig. 5. UV and CD spectra of 0.5 mg/ml IgG2a mAb in 20 mM acetate buffer (contain0.05 cm cell (Jasco 720).
there is an increase in absorbance intensity as evidence ofaromatic residues (Tyr, Tryp and Phe) ionisation, also, the pHcauses a change in the electrostatic interaction. In the far UVspectra, there is a small increase in the absorbance intensity
ing 0.2 mg/ml PS80 and 140 mM NaCl) as a function of pH, 22 °C, 1 cm and
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at pH values of 2.90, 4.10, 9.05, 10.33 and 11.50 with thehighest increase at pH 2.9. The far UV CD shows that theIgG2a mAb retains most of its native secondary structure.
The start of a significant change can be seen as a shift ofthe spectra at pH 4.10, in the far-UV CD spectra, whichbecomes more significant at pH 2.90 showing a broadeningof the β-sheet band at ~217 nm accompanied with enhancednegative CD intensity indicating the beginning of unfoldingand the appearance of a new form of β-sheet. Above pH 5.80,there is an upward shift with the greatest shift at pH 11.50.indicating a conformational change. The solution was turbidand there were visible precipitates indicating aggregationand flocculation which were irreversible after changing thepH back to pH 5.5. In the near UV CD spectra, there isevidence of CIDS at pH 10.33. CIDS is a differential quantityand will not be detected in the UV spectrum, however, it is agood indication of a change in the tertiary structure. Also,while the secondary structure is mostly retained at pH 2.9,there is a significant upward shift at pH 2.9 at ~250–300 nm,which indicates that the native tertiary interactions areabsent (Dolgikh et al., 1981; Ejima et al., 2007). This spectralshift may also suggest differences in disulfide structure(Kosen et al., 1981; Ejima et al., 2007) and that the moleculeis in a compact state termed “molten globule” or “compactintermediate” conformation (Prasad et al., 2012) that aggre-gates and, ultimately, precipitates. However, the presence ofaromatic signals resembling those of the native proteinindicates that at pH 2.9 the protein retains a distinct tertiarystructure and suggests that no gross conformational changehas occurred (Ejima et al., 2007). This may be explained bythe charge distribution on the back bone and the side chainsof the IgG molecules. Electrostatic interaction in proteinmolecules is affected by temperature, pH, ionic strength,size and the concentration of the additives. The electrostaticpotential encountered in IgG depends on the degree ofexposure to the solvent, and the interaction with dipoles inproteins and its interaction with other titrating charges(Mathes and Friess, 2011; Chan et al., 2012; Rayner et al.,2013). The results confirm that under these solutionconditions, a pH range of 5.06–8.0 is a stable pH range forthe IgG2a mAb. The plot of total charges on the antibodyagainst pH (Fig. 6) shows that the most stable pH is 5.5.
0 2 4 6 8 10 12-150
-100
-50
0
50
100
150pH5.5 line
Absolute charge on immunoglobulin
Total charged species
Tot
al c
harg
e on
ant
ibod
y
pH
Fig. 6. The effect of pH on electrostatic interaction in an IgG.
Nonetheless, the calculated net charge is not necessarilyidentical with the actual charge.
3.2.1. In the absence of PS 80The pH range 2.90–9.0 was studied to elucidate the
stabilizing effect of PS 80 at various pH conditions. The pHtitration was performed as indicated before. UV and CDspectra in the far UV region showed no significant changeconfirming the rigidity of the protein backbone (results notshown). However, in the near UV region (Fig. 7), the UVspectra show evidence of turbidity more significantly at pHvalues of 2.95, 8.3 and 9.2. The plot of Asc at 400 nm vs. pH inFig. 7 shows that the most stable pH conditions for the IgG2amAb in the absence of PS 80 are between pH 5.3 and pH 6.5.The CD spectra do not show any significant change except atpH 2.95 where the upward shift is considerably increased.Moreover, there is no significant CIDS and by comparison toFig. 5, the positive feature at ~292 nmbecame sharper andwasblue shifted to ~290 nm. These observations are consistentwith others (Serno et al., 2010), indicating that non-ionicsurfactants stabilize proteins in terms of minimizing proteinaggregation at interfaces due to stresses such as pH, shakingand freeze–thaw.
3.2.2. Additives effects at optimum pH conditionsSince pH 5.5 seems to be optimum to reconcile the aspects
of conformational stability, the effect of stabilizers was inves-tigated at this pH in all cases.
The sodium chloride effect on the IgG2a mAb at pH 5.5 and0.2 mg/ml PS 80 is shown in Fig. 8a and b. In the near UVregion, there is a decrease in absorbance intensity at ~280 nmwith increasing NaCl concentration indicating a change in thearomatic residues 'environment. There is no significant changein the far UV region. The near UV CD spectra exhibit a change inthe feature at ~292 nm, again indicating a change in the Trypenvironment. Also, there is an upward shift with increasingNaCl concentration between 290 nm and 240 nm indicatingconformational change. At low salt concentrations, there is noevidence of turbidity nor CIDS. This could be due to the socalled “salting-in” effect, as the addition of salt can increase themagnitude of favourable interactions between charged proteinresidues and their image charges in the high dielectric solventleading to an enhancement in protein solubility as the saltconcentration is sufficiently increased.
The effect of high salt concentration (above 2.0 M) on theIgG can be seen in the CD spectra above 300 nm, where smallevidence of CIDS are apparent, which most likely representconformational change. mAbs are composed of multipledomains, each of which may be susceptible to unfoldingthat may or may not reveal aggregation prone regions orso-called “hot spots” (Ejima et al., 2007). This may explainthe low turbidity in the UV spectra at high salt concentra-tions. However, the onset of aggregation at 4.0 M saltconcentration which is evident in the small turbidity in theUV spectra and the CIDS in the CD spectra could be due tounfavourable interactions. These unfavourable interactionscan be minimized by protein unfolding, aggregation andprecipitation, thus causing the protein molecules to always“salt-out” of solution at high enough salt concentrations.Also, at higher ionic strength, electrostatic repulsions be-tween aggregates may not be sufficient to prevent them from
-0.00015
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-0.00005
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1.2
2 3 4 5 6 7 8 9 100.00
0.03
0.06
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0.12
0.15 Turbidity in the absence of PS 80
pH 8.3
pH 9.2pH 2.95
Wavelength (nm)
TurbiditypH 8.3
pH 9.2
pH 2.95
Abs
orba
nce
A S
c 400
nm
pH
240 260 280 300 320 340 360 380 400
240 260 280 300 320 340 360 380 400
ΔA
Fig. 7. UV and CD spectra of 0.5 mg/ml IgG2a mAb in 20 mM acetate buffer containing 140 mM NaCl (no PS80) as a function of pH, at 22 °C, 1 cm.
0.0 M NaCl 1.0 M NaCl 2.0 M NaCl 3.0 M NaCl 4.0 M NaCl
Increasing NaCl conc
Wavelength (nm)
Abs
λ 2
80
NaCl Conc. (M)
Abs λ 280
Abs
λ 2
80
PS80 Conc. mg/ml
Abs λ 250
Abs
λ 2
50
360340320300280260240
360340320300280260240200 360340300280260240220
320200 360340300280260240220
ΔAA
bsor
banc
e
Fig. 8. UV and CD spectra of IgG2a mAb showing (a) the effect of various concentrations of NaCl on the absorbance spectra and a binding curve of NaCl conc. vs.IgG2a absorbance at λ 280 nm (b) the effect of NaCl on the CD spectra (c) the effect of various concentrations of PS 80 on the absorbance spectra and a plot ofPS80 concentration vs. absorbance maxima at λ 280 and λ 250 (d) the effect of PS80 on the CD spectra.
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coalescing and ultimately forming macroscopic particles orprecipitates (Thakkar et al., 2013). On the other hand, NaCldid not induce significant changes to the secondary structureof IgG (Fig. 8b). However, NaCl effects can be represented inthe binding curve (Fig. 8a) which shows a decrease ofabsorbancewith increasing salt concentration till it reaches thepoint where further increases in the salt concentration wouldlead to the onset of unfolding and aggregation, as is evident inboth the UV and CD spectra in the near UV region, suggestingthat the salt most probably have decreased mAb solubility,presumably by screening the repulsive surface charge of themAb present (Banks et al., 2012).
The PS 80 effect on the IgG2a mAb at pH 5.5 and 140 mMNaCl is shown in Fig. 8c and d. In the far-UV region, there ishardly any measurable difference in the UV and CD spectraupon the addition of PS 80 indicating the rigidity of thesecondary structure (results not shown). In the near UVregion, the UV absorbance spectra exhibit an increase inintensity with increasing PS80 concentration particularly at260–240 nm, indicating a significant effect on tertiary struc-ture (aromatic residues). Under these conditions, the increasein intensity suggests a stabilising effect of PS80 at the air-waterinterface. However, at a certain critical PS80 concentration(CPC), the effect reached a plateau (Fig. 8c and d) at whichpoint, the PS80 concentration is independent of the proteinconcentration. The plot of absorbance at 280 nm and 250 nmvs. PS80 concentration (Fig. 8c), shows that at 280 nm whichmainly represents Tyr and Tryp, the highest increase inabsorbance corresponds to 0.2 mg/ml PS 80, indicating
0.0
0.4
0.8
1.2
-0.00020
-0.00015
-0.00010
-0.00005
0.00000
0.00005
Dec
reas
ing
Turb
idity
Far from detector ASc far from detector ASc Midway Midway ASc near detector Near the detector
a Solvent corrected - Turbidity not corrected
Abs
orba
nce
Incr
easi
ng C
IDS
Far from detector Midway Near the detector
Solvent correctedc
Wavelength (nm)
Wavelength (nm)
240 280 320 360 400
240 280 320 360 400
ΔA
Fig. 9. Turbidity measurements of IgG2a mAb in 20 mM acetate buffer (containingpH 9.2 showing the simultaneous measurement of turbidity where the cell is at dspectrum at 400 nm which is used for turbidity correction. (b) UV spectra correcteCD. (d) a plot of cell position from the detector vs. turbidity at 400 nm.
increased solubility of mAb at this concentration. On theother hand, the plot at 250 nm shows increased intensity withincreasing PS80 concentration going towards the CPC pointwhere PS80 concentration is independent of the proteinconcentration. The UV spectra also show no evidence ofturbidity at 0.2 mg/ml PS 80, indicating that 0.2 mg/ml ofPS 80 is the most stable concentration.
The results indicate that the additives tested most probablyinfluence the mAb association rate by virtue of their effect onthe solubility of the native state at room temperature, ratherthan by destabilizing partially unfolded intermediate state(s)that lead to aggregation and precipitation (Banks et al., 2012).Moreover, the additives involved in this study did not sig-nificantly affect the conformational stability of mAb, however,they were able to prevent aggregation. This is most probablydue to an increase in the hydrophilicity of the protein surface,because their affinity for water is greater than that for theco-solvent due to preferential hydration (Shimizu and Smith,2004; Szenczi et al., 2006), which is clearly reflected in thenear-UV-CD spectra (Fig. 8).
3.2.3. Aggregation (turbidity) measurementsThe aggregation behaviour of the IgG2a mAb can be ad-
dressed using the simultaneous measurements of turbidity(light scattering), this in turn can be achieved by measuringthe absorbance values at a wavelength where the moleculedoes not normally absorb (as described earlier). The pHinduced aggregation behaviour of the mAb (at pH 9.2) wasmeasured at 400 nm. In addition, the effect of the distance
0.0
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0.8
4 8 12 16 24
0.04
0.08
0.12
b Solvent corrected - Turbidity corrected
Abs
orba
nce
Wavelength (nm)d
Distance from detector (cm)
240 260 280 300 320 340 360 380 400
20
Asc
λ40
0
0.2 mg/ml PS80 and 140 mM NaCl) at 22 °C. (a) UV spectra of IgG2a mAb atifferent distances from the detector. Asc denotes the extrapolated scatteringd for turbidity. (c) CD spectra showing CIDS and the effect of cell position on
163A. Aboel Dahab, D. El-Hag / Journal of Immunological Methods 405 (2014) 154–166
between the sample and the detector on the observedturbidity was investigated in order to properly monitorthe mAb aggregation rate. Fig. 9a shows the UV spectra ofthe mAb at pH 9.2 measured at different distances from thedetector. The mAb aggregation is apparent, however, thedistance between the sample and the detector has a significanteffect on the measured turbidity. The measured turbidity isdirectly proportional to the distance from the detector (Fig. 9d).As turbidity is related to fraction mAb aggregated, thus, theposition of the cell from the detector is pivotal to achievingaccurate results in aggregation measurements. This is confirmedby correcting the spectra for turbidity (as described earlier) asshown in Fig. 9b, where it is apparent that all spectra arecompletely overlapped after correction. Consequently, taking theresults of turbidity for the midway and far from the detectorwould give a false indication of the fraction mAb aggregated,which would affect aggregation kinetic measurements. On theother hand, Fig. 8c shows the corresponding CD spectra, where itis apparent that CIDs are inversely proportional to thedistance of the sample from the detector. This can be ex-plained by difference between turbidity measurements inthe UV and CD spectra. Turbidity measurements in UV ismerely a direct measure of the total scattering spectra, whilein CD it is a differential quantity measuring the difference ofscattering between left and right circularly polarised light.
3.3. Thermal stability
Equilibrium thermodynamic analysis of protein unfoldingis often hampered by its irreversibility, which usually results
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a
CD
(m
d e
g)
85 oC
20 oC
c
CD
m d
e g
Wavelength (nm)
T 20 oC T 68 oC T 73 oC T 78 oC T 85 oC
Tm= 60 oC
Temperature (K)
200 220 240 260 280 300 320 340 360
200 220 240 260 280 300 320 340 360
280 300 320 340 360
ΔA λ
216
Fig. 10. CD spectra of IgG2a mAb in 20 mM acetate buffer pH 5.5 containing 0.2 mg/mof variable temperature (20–85 °C) and the melting curve. (b) shows the toleratedabove the melting temperature. (d) shows irreversible denaturation after cooling b
from aggregation of thermally denatured protein. However, aconvenient method to monitor in real time protein aggrega-tion during thermal folding/unfolding transition is to recordstructural changes and CIDS data in circular dichroism (CD)experiments. Fig. 10a shows the effect of temperature(20–85 °C) on the CD spectra of IgG2a mAb in the nearand far UV regions. The mAb is fairly stable at temperaturesup to 60 °C with the onset of aggregation apparent at 65 °C(Fig. 10b), however, in the near UV region, the aromaticresidues' region shows a compact structure (molten globule) at65 °C. At higher temperatures (Fig. 10c), the CD band at~218 nm is decreased in intensity and shifted towards longerwavelengths due to high mobility of the protein with in-creasing temperature. Above 73 °C, the CD is substantiallyreduced to a broad negative band at ~224 nm, in addition tothe upward shift.
In the near UV region, the CD of the tryptophan specificfeature represented by a wavelength ~292 nm is relativelyinsensitive to temperature up to 60 °C indicating that tryptophanmobility is low. Above 65 °C, the positive CD band at ~292 nm(tryptophan) is lost, the IgG molecule begins unfolding and thenear UV CD effectively collapses as the antibody aggregatesfurther and precipitates, which is indicated by CIDs and visualobservation.
Classically, at high temperature, as a folded protein is heated,it starts to unfold. However, as the temperature passes through68 °C to 73 °C, rather than simply unfolding, the results in thefar UV region indicate that the IgG exhibits a change in β-sheettype and a long wavelength shift of the negative band at~218 nm CD. This suggests that the first unfolded state was
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0
1
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3
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5
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0
5
CD
(M
d e
g)
Temperature (oC )
CD 274 nm CD 292 nm CD 257 nm
b
T 20.6 oC T 29.4 oC T 39.7 oC T 49.3 oC T 53.9 oC T 59.6 oC T 65.0 oC
CD
(m
d e
g)
After cooling back to 20 oC(Irreversible)
d
CD
(m
d e
g)
Wavelength (nm)
200 220 240 260 280 300 320 340 360
200 220 240 260 280 300 320 340 360
20 30 40 50 60 70 80
l PS80 and 140 mMNaCl in the near and far UV regions. (a) shows the effecttemperature range. (c) shows comparison between temperatures below andack to 20 °C and plots of various wavelengths vs. temperature.
164 A. Aboel Dahab, D. El-Hag / Journal of Immunological Methods 405 (2014) 154–166
transient leading to the formation of a new β′-sheet state. Athigher temperatures, at no point was there any evidence of acontribution from a negative CD centered at 200 nm, whichwould be indicative of the presence of a disorder state(random coil). Therefore, the negative CD at ~224 nm isrepresentative of a third β′′-sheet state. This indicates thatthe other forms of β-sheet are a transient states that lead toaggregation and precipitation at higher temperatures.Heating the antibody to ~85 °C is irreversible and thethree β-sheet states can be identified as:
Aobs. = AU + AF, where Au and AF are the absorbance ofthe unfolded and folded state respectively. Then
K ¼ UF: ð1Þ
The curve analysis is based on Van’t Hoff ‘s isochoreequation:
logK ¼ −ΔHRT
ð2Þ
For the temperature plot (Fig. 10a) and the calculation ofthe temperature midpoint of a thermal unfolding (melting)transition (Tm), the equation is derived and re-written to beused in Origin Microcal computer programme as:
The temperature curve (Fig. 10a) which is yielded byplotting temperature vs. ΔΑ at λ216 shows that Tm is ~60 °Cand the onset temperature of thermal unfolding (T0) is~53 °C. Fig. 10d shows the CD spectra of IgG2a mAb in thenear and far UV regions after cooling from 85 to 20 °C. Theirregular structure of the IgG molecule, which does not fit a
Native CD
Native
K1
k 2
K2
CIDs and
Aggre
Unfold
CD chang
Slight CD change
Compact
Fig. 11. Aggregation kinetics of IgG2a mAb by CD, where K1 is the equilibrium consprotein, K2 is the equilibrium constant for association of native structure, and k2 is
random coil spectrum is indicative of irreversible unfoldingas a result of heat denaturation. Plotting temperature vs. CDfor various wavelengths (Fig. 10d) shows that conforma-tional stability is fairly maintained at temperatures up to65 °C. Collectively, the results demonstrate that a temper-ature region up to 65 °C under these conditions is the moststable region for this antibody in terms of conformationalstability and thermally induced aggregation behaviour.
However, below 60 °C there was visible evidence of aggre-gation, although insignificant, it indicates that aggregation canoccur from the association of either the unfolded or the nativestate of IgG. This confirms that heat aggregation of IgG occurs viaboth native and unfolded stateswith different rates for each casedepending on the rigidity of the IgG structure, excipients, pH andtemperature. Therefore, the effect of temperature on theequilibrium constant of unfolding and association can bedepicted as in Fig. 11.
Increased melting temperature typically translates to a shiftin the equilibriumconstant of unfolding towards the native state,i.e., decrease in K1. Thus, there will be a reduction in the pop-ulation of unfolded protein leading to aggregation. However, theprotein stabilizing excipients can enhance self-association, i.e.,greater k1, indicating that they may enhance aggregation evenwhen there is a paucity of unfolded proteins. The stabilizingexcipients can also increase the equilibrium constant, K2 ofself-association of the native state. Aggregation often becomesirreversible as the extent of self-association increases. However,as long as such self-association is reversible, it causes no damageto the protein.
4. Conclusion
Analysis of the CD spectra reveals that the β-sheet is themost dominant secondary structure element present in thisIgG, indicating that it belongs to the all-β IgG sub-class. Themost crucial part in pH titration is avoiding “shocking” theantibody and prematurely inducing aggregation due to highinitial pH or additive concentrations during the addition of adroplet of titre. The most convenient method is the gentlemulti-pot pH titration. This methodology provides an insightinto the complex mechanism controlling aggregation of such
k2
and CD collapse
CD collapse
Aggregated
gated
ed
e CIDs
tant for folding/unfolding, k1 is the rate constant for association of unfoldedthe rate constant for association of native protein oligomers.
165A. Aboel Dahab, D. El-Hag / Journal of Immunological Methods 405 (2014) 154–166
multi-domain proteins as IgG antibodies. It is evident thatIgG2a are less stable and aggregate more readily at pH b 3.5in the absence of PS80. The secondary structure showedsignificant conformational stability at all pH values indicat-ing that acid-induced aggregation of typical IgG2s proceedspredominantly via tertiary structure-related aggregationpathway. The stabilisation effect of PS80 is mainly preventingsurface denaturation, reducing the environment effect on thearomatic residues which are more exposed to the solvent. Thiscan explain the better stability against pH variations. As theinclusion of NaCl did not induce any significant changes to thesecondary structure of IgG, the stabilization/destabilisationeffect of the salt can be attributed to its effect on electrostaticrepulsive forces, which have an effect on protein solubility(Banks et al., 2012). UV spectrum can readily be corrected forlight scattering using λ-n corrected absorption to estimate theamount of spectroscopically active material. This indicates thatthe geometry of light detection is important and that UVmeasurements may vary from instrument to instrument.
Study of the temperature variations of the CD spectrum ofthe antibody reveals complexity. It showed that the proteindoes not change conformation appreciably across the tem-perature range of study up to 65 °C. However, the CD spectra(73 °C–85 °C) are not indicative of any simple events andprobably represent precipitation and flocculation. The inter-pretation of its stability solely from the melting data isdifficult, as it depends on the rate of aggregation as well as onthe thermodynamic stability of the protein. In this case, theonset temperature of thermal unfolding T0, may be moremeaningful, as it is affected less by the aggregation process.Several processes can potentially be monitored which inturn will provide elegant methods to monitor the effects ofprotein environment and additives. Aggregates of therapeu-tic proteins can increase the likelihood of adverse immuno-genic effects during therapy, which has been linked toincreased patient morbidity or mortality (Latypov et al., 2012).In the process of heating a protein (antibody) solution, it isimportant to distinguish between unfolded, refolded, aggre-gated, and flocculated states. The simultaneous monitoring ofabsorption, turbidity and optical activity provides informationabout these states and helps assign aggregated states. It alsoprovides important criteria for reliable CD measurement froman instrumental point-of-view and is an important feature of aprotocol for CD measurements. Simultaneous monitoring ofseveral optical parameters of the same sample on the sameinstrument is not only time saving but also ensures that anyprocesses occurring in solution are monitored under the sameconditions. Concentration variations can be monitored to betterensure that CD variations reflect conformational changes moreprecisely. An important factor (the subject of future work) thatshould be realised at the practical formulation and storage ofmAb is the effect of protein concentration onmAb dimerization,which is supposed to be controlled by the colloidal stability ofthe native state that is believed to be followed by a largelyirreversible conformational change to the non-native dimer,given the lack of appreciable disassociation of this species upondilution (Thakkar et al., 2013). Also, factors that affect thereliability of results such as the robustness of the technique,noise, time scale of data collection (cf. time constant), spectrumscan speed, and spectral bandwidth will be the subject of afuture publication.
Finally, it is important to note that most proteins functionunder physiological conditions, and at this pH and salt con-centration, some proteins are largely destabilized. Hence,stability and function do not correlate. Moreover, stabilitymay disfavour functionality and a too stable protein does notfunction as well in the cell. However, avoidance of aggre-gation is especially critical in a cellular environment with ahigh protein concentration. Only antibodies give CIDS. Thispromotes the need for 90° detection for simultaneous lightscattering detection and measurements starting from 400,500 or 600 nm.
Conflict of interest
The authors declares no conflict of interest.
Acknowledgements
We thank Pfizer PLC and Applied Photo Physics (APL) fortheir financial and scientific support with regard to materialsand instrumentations.
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