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Available online at www.sciencedirect.com

Journal of Chromatography A, 1173 (2007) 159–164

Characterization of protein conjugates using capillary electrophoresis

Samir Safi, Zouhair Asfari, Agnes Hagege ∗Laboratoire de Chimie Analytique et Minerale, IPHC-DSA (UMR 7178), ULP, CNRS, ECPM,

25 rue Becquerel, F-67087 Strasbourg Cedex, France

Received 18 May 2007; received in revised form 4 October 2007; accepted 8 October 2007Available online 12 October 2007

bstract

With the aim of generating antibodies, a calix[4]arene-crown-6 was coupled to bovine serum albumin. For that purpose, a complete procedure

o optimize and characterize the coupling of hydrophobic haptens based on capillary electrophoresis (CE) was developed. We demonstrated thexistence of a polynomial relationship between the electrophoretic mobility (μep) and the hapten density. This correlation was used not only totudy the coupling reaction in terms of optimization and kinetics but also to determine the average coupling molar ratio of any given conjugate.n estimation of the heterogeneity of these conjugates by simulation of experimental peaks was also proposed. 2007 Elsevier B.V. All rights reserved.

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eywords: Capillary electrophoresis; Conjugation; Hapten; Bovine serum albu

. Introduction

The use of protein conjugates has been well described inhe literature [1–5]. Many methods were then developed toetermine the coupling density which can affect both the speci-city and quantity of antibodies [6,7]. These methods includeeparative methods such as size-exclusion chromatography [8]r sodium dodecyl sulfate polyacrylamide gel electrophore-is (SDS-PAGE) [9] and non-separative ones. Since direct UVeasurements can only be applied when a component of the

onjugate has a unique absorbance [10], trinitrobenzenesulfoniccid derivatization (TNBS) [11] is the most commonly usedethod. Radiolabelled haptens were also used [12] but requiredsignificant synthetic effort. Time-of-flight mass spectrometrysing matrix assisted laser desorption ionization (MALDI-TOF-S) [13] has been shown to provide satisfactory results for the

etermination of the coupling ratio of conjugates.However, all these methods were used to determine the aver-

ge number of haptens per carrier protein. In order to understandhe behaviour of protein conjugates, it is necessary to know the

xact nature of the conjugates, i.e. their stoichiometry and theireterogeneity. Recently, capillary electrophoresis was showno be efficient in studies of the formation of hapten-protein

∗ Corresponding author. Tel.: +33 3 90 24 27 27.E-mail address: hagegea@ecpm.u-strasbg.fr (A. Hagege).

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021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2007.10.018

onjugate heterogeneity

onjugates for unlabelled compounds or non-UV-absorbingompounds [14,15].

In this study, capillary electrophoresis will be proved toe a fast tool which can provide numerous informations inhe field of protein conjugates synthesis. We have previouslyeported the synthesis and characterization by MS and TNBSf calix[4]arene-crown-6 based immunogens for a potential usen the immunoanalysis of cesium ions [16]. The coupling reac-ion will be monitored through the variations in electrophoreticobilities. Protein conjugates will also be characterized in terms

f hapten density and heterogeneity.

. Experimental

.1. Chemicals and reagents

Bovine serum albumin (BSA) and trinitrobenzenesulfoniccid (TNBS), dimethylformamide (DMF) and HCl were pur-hased from Sigma–Aldrich (Saint Quentin, France). Sodiumodecyl sulfate (SDS) and glycine were purchased from MerckFontenay-sous-Bois, France).

1-(N-succinimide-5-oxavalerate), 3-methoxycalix[4]arene-rown-6 (NHS-calixarene) was synthesized according to the

rocedure previously described [16].

Na2B4O7·10H2O, used for buffer preparation, was purchasedrom Merck and daily prepared in ultrapure Milli-Q water andltered using a 0.45 �m Analypore filter (Elancourt, France).

1 ogr. A 1173 (2007) 159–164

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60 S. Safi et al. / J. Chromat

used silica capillaries were purchased from Thermo Electronorporation (Thermo Electron, Courtaboeuf, France). Methanolnd NaOH purchased from Acros (Noisy Le Grand, France)ere used for the CE capillary conditioning.

.2. Coupling of NHS-calix[4]arene to BSA

To 1 mL of a BSA solution (4 mg/mL, 96%) in a 25 mModium borate buffer at pH 9.2 was added a DMF solu-ion containing the NHS-calixarene at various concentrations5–25 mg/mL). The volume of these DMF solutions was variedrom 30 to 100 �L. The mixtures were vortexed and incu-ated overnight at 4 ◦C. These solutions were then storedt 2 ◦C without purification. Each conjugation was done inuplicate.

For kinetics studies the coupling mixtures of conjugates wereirectly prepared in the electrophoresis vials.

.3. Spectrophotometric measurements

A modified version of Habeeb’s TNBS assay [11] was used.wenty-microliter aliquots of the crude mixtures were diluted0 times in a 0.1 M sodium borate buffer pH 9.2. Standard solu-ions of glycine (2–9 �g/mL) in a 0.1 M sodium borate bufferere also prepared. To 1 mL of the conjugation mixture or thelycine standards were added 500 �L of a 0.01% TNBS in a.1 M sodium borate buffer and incubation was performed at2 ◦C for 2 h. Five hundred microliters of 10% (w/w) SDS and50 �L of 1 M HCl were then added and the absorbance waseasured at 335 nm using an Agilent spectrophotometer, modelP8453 (Massy, France).

.4. Capillary electrophoresis measurements

All experiments were performed with a Beckman P/ACEDQ apparatus (Roissy Charles de Gaulle, France) controlled

y a computer using the 32 karat software.Samples were injected for 3 s at 1.38 kPa. For all separations,

xcept for conjugate recovery experiments, a 30 cm × 75 �m.D. fused silica capillary was used for separations. Betweenuns, the capillary was flushed at 138 kPa with 0.1 M NaOHor 1 min and running buffer for 3 min. Separations were real-zed using a 25 mM borate buffer pH 9.2 at 12 kV and 25 ◦C.onjugate recoveries were determined using a single capillary

ecut to obtain different total lengths ranging from 30 to 70 cm,eparation lengths varying from 20 to 60 cm. Separations wereerformed using a 25 mM borate buffer pH 9.2 at 25 ◦C under anlectric field of 400 V cm−1. Recovery rates were determined byaking the corrected surface area obtained on the 30 cm-capillarys reference after taking into accounts differences in injectiony using the calixarene under its carboxylate form as internal

tandard.

Detection was performed in UV between 200 and 300 nmsing a diode array detector. For that purpose, a detection win-ow was created at 10 cm from the cathodic end of the capillaryy simply removing the polyimide coating.

m

μ

Fig. 1. Schematic representation of the synthesized conjugates.

. Results and discussion

.1. Correlation of the coupling process on electrophoreticobility

BSA conjugates were obtained by reaction of succin-midyl ester calix[4]arene-crown-6 with the NH2-groups ofysine which leads to stable peptidic bonds. For that pur-ose, solutions were prepared at different coupling molaratios (NHS-calixarene:BSA) using 100 �L of DMF contain-ng the NHS-calixarene at various concentrations. The obtainedonjugates, schematically represented in Fig. 1, were calledonjugates 1–10. The initial NHS-calixarene:BSA ratio wasradually increased going from conjugate 1 to conjugate 10. Thelectropherograms resulting from the separation of some of theseonjugates mixtures are represented in Fig. 2. In addition to theSA peak, the electropherograms show the appearance of 2 otherompounds, i.e. calixarene under its carboxylate form resultingrom the hydrolysis of the non-reacting NHS-calixarene and N-ydroxysuccinimide (NHS) resulting from both hydrolysis andonjugation.

A shift of the BSA peak was observed and attributed to thencrease of the global negative charge of the conjugates becausef the coupling through the amine functions. Moreover, a pro-ressive widening and flattening due to the coupled calixarenesistribution in the formed conjugates was noticed.

In similar studies using a hydrophilic hapten, Pedron et al.15] correlated the observed electrophoretic mobility to the cou-ling density using a polynomial regression (magnitude 3). Inrder to verify this correlation in the case of hydrophobic hap-ens, the obtained electrophoretic mobilities were related to their

olar coupling density determined by the TNBS method.For all the conjugates, the electrophoretic mobility was deter-

ined using the following equation:

ep = LtLd

V

(1

t− 1

teo

)(1)

S. Safi et al. / J. Chromatogr. A 1173 (2007) 159–164 161

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cThe influence of the initial number of haptens was inves-

tigated. For that purpose, three solutions were prepared in a5% DMF solution using various NHS-calixarene:BSA ratios.Aliquots were analyzed every 10 min for 5 h. Fig. 4 displays the

ig. 2. Electrophoregrams of the conjugation media as function of the added ame) conjugate 8. Separation conditions: fused silica capillary of 30 cm × 75 �ms. Detection: UV at 200 nm 1: BSA or BSA conjugate, 2: DMF, 3: calixarene

here μep is the electrophoretic mobility of the solute, Lt is theotal length of the capillary while Ld is the length to the detec-ion window, V is the applied voltage, t is the migration timef the conjugate and teo is the migration time of the electroos-otic marker (DMF). The electroosmotic flow was measured at

20 nm from the migration time of the DMF contained in theamples.

First of all, the shift of the calixarene–BSA peak was corre-ated to the coupling ratio, each value of μep being calculateds the average of three experimental measurements. The stan-ard deviation was found to be less than 2 × 10−6 cm2 V−1 s−1.sing the TNBS method, the determination of the averageumber of coupled calixarenes was determined from five mea-urements and the standard deviation was found to be 1.5. Theorrelation between these data is illustrated in Fig. 3.

A polynomial regression (magnitude 3) with a correlationoefficient of 0.999 was determined:

= 3.14 × 10−5x3 + 8.49 × 10−4x2

+ 1.54 × 10−1x + 15.2 (2)

sing this equation, the average molar coupling density of cal-xarenes on the BSA protein can be rapidly assessed from thelectrophoretic mobility of the observed conjugate peak.

Fct

of NHS-calixarene (a) BSA; (b) conjugate 1; (c) conjugate 4; (d) conjugate 6;electrolyte: 25 mM borate pH 9.2; V = 12 kV and T = 20 ◦C. Injection: 0.2 psi,its carboxylate form, and 4: NHS.

.2. Kinetics studies of the conjugation process

Due to the short analysis time and the small sample volumes,apillary electrophoresis allows kinetics studies.

ig. 3. Correlation between the electrophoretic mobility of calixarene–BSAonjugates and the average number of calixarenes determined by spectropho-ometry after TNBS derivatization.

162 S. Safi et al. / J. Chromatogr. A

Fig. 4. Influence of the incubation time on the electrophoretic mobility of thec2

er

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onjugates for different initial NHS-calixarene:BSA molar ratios: (©) conjugate; (×) conjugate 7; (�) conjugate 9.

lectrophoretic mobilities of the conjugates obtained in a singleun as a function of the reaction time.

The coupling process seems to be a two-step mechanism. Inhe first step, the electrophoretic mobility increased till around0 min to finally reach a plateau. For the highest initial NHS-alixarene:BSA ratios, a second step appeared, characterizedy a further increase in electrophoretic mobility till a secondlateau. It seems that the NHS-calixarene:BSA ratio rather gov-rns the first step. Indeed, the higher NHS-calixarene:BSA ratioas, the shorter the first plateau was. It can also be noted that

or the weakest NHS-calixarene:BSA molar ratio, the durationf the first step exceeded 5 h and no further increase of the μepas observed. The number of conjugated calixarenes was cal-

ulated from Eq. (2) by using the μep measured at the end of theonjugation experiment. This calculation led to 13, 28 and 30or conjugates 2, 7 and 9, respectively.

The coupling of hydrophobic haptens also requires the usef organic solvents. However, to our knowledge, no studiesetermining the influence of the organic solvent volume wereeported. For that purpose, four conjugates were prepared at dif-erent DMF ratios (3, 5, 10 and 20%) by adding NHS-calixareneolutions at the desired concentration to obtain identical initialHS-calixarene:BSA molar ratios of 50:1. Fig. 5 displays the

lectrophoretic mobilities of the conjugates obtained in a sin-

le run as a function of the reaction time for different DMFercentages.

ig. 5. Influence of the incubation time on the electrophoretic mobility of theonjugates for different DMF (v/v) percentage: (©) 3%; (×) 5%; (�) 10%; (�)0%.

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1173 (2007) 159–164

An increasing percentage of DMF was shown to decreasehe velocity of the coupling, mainly by increasing the first stepuration. When using 5 and 10% DMF, the final coupling den-ity was found to be nearly the same (30:1). For the 20% DMFixture, the final coupling density was smaller (23:1). It can be

ssumed that the slow conjugation velocity favors the hydrolysisf the NHS-calixarene, which cannot react anymore with BSA.

In the case of 3% DMF, the first step was slower thanxpected. This can be explained by an insufficient solubil-ty of the NHS-calixarene in the conjugation medium. Thisssumption is reinforced by the appearance of a precipitate andubsequently a lower coupling ratio (23:1).

.3. Assessment of the coupling heterogeneity

A progressive peak widening was also noticed as more cal-xarenes were being coupled.

Since the main factors that contribute to peak broadeningre longitudinal diffusion, injection and adsorption, the totalariance can therefore be expressed as:

2tot = σ2

diff + σ2ads + σ2

others (3)

here σ2diff and σ2

ads are the variances caused by longitudinaliffusion and wall adsorption, respectively and σ2

others representshe variances due to injection, detection, conductivity effects,tc. [17,18].

In order to verify that the coupling does not favor interactionsith the inner surface of the capillary, separations of a highly

oupled BSA conjugate, labelled 9 (presenting an average num-er of coupled calixarenes of 30) were performed under differentoltages and separations were carried out without any flushingf the capillary between runs and the electroosmotic mobilityas measured.The average electroosmotic velocity was found to be

5.537 ± 0.006) × 10−4 cm2 V−1 s−1 suggesting that no alter-tion of the capillary surface occurred.

Moreover, the adsorption was also examined using the pro-edure described by Towns and Regnier [19] and Ghosal etl. [20,21]. Results of separations of conjugate 9 performed inriplicate for different capillary lengths are presented in Fig. 6.

Results show that migration times of both neutral marker androtein conjugate increased linearly with the capillary length.hese data suggest once again that no alteration of the capillaryurface occurred. Moreover, the recovery rate of the conjugatealculated as described in Section 2 was shown to be constantnd equal to 100%.

As a consequence, adsorption phenomena if any can beeglected and the peak widening of a single conjugate (σ2

tot)an be expressed as:

2tot = σ2

diff + σ2others = 2Dmt + σ2

others (4)

here all the variances are expressed in length units, Dm is the

iffusion coefficient of the solute and t, its migration time.

To determine the apparent diffusion coefficient of BSA, wesed the stopped migration method described by Righetti et al.22]. BSA was first injected and separated using the conditions

S. Safi et al. / J. Chromatogr. A 1173 (2007) 159–164 163

Fig. 6. Influence of the capillary length on the conjugate recovery rate ( )and the migration times of both neutral marker (©) and conjugate (×). Separa-tp2

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ion conditions: fused silica capillary of 75 �m I.D.; electrolyte: 25 mM borateH 9.2; E = 400 V cm−1 and T = 20 ◦C. Injection: 0.2 psi, 3 s. Detection: UV at00 nm.

dopted for the conjugates analysis. In the following runs, BSAas separated till it reached approximately the middle of the

apillary and the sample was let to diffuse in the absence ofn electric field during times varying from 600 to 6900 s. Thelectric field was then applied till BSA reached the detector. Theotal variance of each peak was then measured and the differenceσ2

tot was calculated as:

σ2tot = σ2

tot(tdiff) − σ2tot(tdiff = 0) (5)

here σ2tot (tdiff = 0) is the variance of the BSA peak obtained

n the first run and σ2tot(tdiff) is the variance of the BSA peak

easured when the analyte was let to diffuse during a durationdiff.

The plot of �σ2tot as a function of tdiff was found to be a

traight line and the diffusion coefficient was then obtainedrom the slope (1.83 × 10−6 cm2 s−1) and found to be equal to.9 × 10−7 cm2 s−1.

As a consequence, σ2tot can be mainly attributed to σ2

othersnd is identical for each conjugate if expressed in length units.2others can be calculated from the BSA peak (Fig. 7(a)) and the

heoretical σ2tot expected for all the conjugates can be calculated

rom Eq. (4) and converted in time units using their migrationimes according to the following equation:

2tot(s

2) = σ2tot(cm2) × t2

L2d

(6)

t will then reflect the artificial widening of the conjugates dueo the increase of their migration times.

All the calculations were performed using the SigmaPlot soft-are (version 8.0). As shown in Fig. 7(a), a Gaussian fit can besed to satisfactorily describe the BSA experimental peak. The σ

alue determined from this fit (σ = 4.36 s) was used to determine2others.

Table 1 presents a comparison of the theoretical σ2tot cal-

ulated using σ2others as well as the σ2

tot calculated using the

eak width (corresponding to 4σ for Gaussian peaks) for theifferent conjugates synthesized. These results reveal that theispersion factors were not sufficient enough to explain theeak wideness. As a consequence, it can be assumed that

wi

w

y a Gaussian distribution (—) (a) BSA; (b) conjugate 1; (c) conjugate 4; (d)onjugate 6. n is the number of attached haptens.

he peak widening reflects a significant increase in conjugateseterogeneity.

Under this assumption, a BSA with n attached haptens shoulde a Gaussian shifted peak corresponding to a single conjugateith a σ(n) calculated from Eq. (6) and an intensity, function of

ts abundance a(n).Attempts to assess the heterogeneity by peak deconvolution

ere previously described in mass spectrometry [23,24].

164 S. Safi et al. / J. Chromatogr. A 1173 (2007) 159–164

Table 1Comparison of σ2

tot calculated from σ2others and from conjugates peak widths

Compound Migration time (s) σ2tot calculated from peak width (s2) σ2

tot calculated from σ2others (s2)

BSA 152.5 19.00 –Conjugate 1 157.4 21.62 20.25C 9C 2

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onjugate 4 162.3 30.6onjugate 6 175.3 66.4

In a similar way, an estimation of the coupling ratioistribution can therefore be achieved by deconvoluting thexperimental peak into a series of consecutive Gaussian contri-utions and so their abundance an can be simulated and adjustedutomatically by the SigmaPlot software to get the best fit withnon-linear regression coefficient of not less than 0.998 [24].he results of these deconvolutions obtained for the conjugates, 4 and 6, presenting an average number of 9, 17 and 24 bondedalixarenes, respectively, are presented in Fig. 7(b–d).

It can be clearly seen that the heterogeneity increases with theolar coupling ratio. The use of a initial small amount of NHS-

alixarenes (conjugate 1) allows the synthesis of conjugatesith a rather homogeneous coupling extent. On the contrary,

n increase of this amount (conjugate 6) leads to the formationf conjugates presenting about 15 different coupling extents.

. Conclusion

Capillary electrophoresis was proved to be an efficient toolo optimize and characterize hydrophobic hapten coupling. Itas used to optimize calixarene coupling conditions (DMF

nd NHS-calixarene:BSA ratio). Kinetic studies gave a furthernsight into the coupling process and the limiting factors weredentified. Capillary electrophoresis was also used for the rapidharacterization of conjugates directly from the coupling mix-ure. An estimation of both hapten density and heterogeneityas proposed.Further studies will be performed to generate antibodies

irected towards cesium using these immunogens. Experimentso incorporate cesium and determine the number of ions carriedy the conjugates are currently in progress.

cknowledgement

This work was supported by a grant to S.S. from the Frenchinistry of Research and Higher Education, Paris, France.

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