Antibody–Ligand Interactions for Hydrophobic Charge-Induction

Article pubs.acs.org/Langmuir

Antibody−Ligand Interactions for Hydrophobic Charge-Induction Chromatography: A Surface Plasmon Resonance Study Fang Cheng,*,†,‡ Ming-Yang Li,†,§ Han-Qi Wang,†,‡ Dong-Qiang Lin,∥ and Jing-Ping Qu† †

State Key Laboratory of Fine Chemicals and ‡School of Pharmaceutical Science and Technology, Dalian University of Technology, Dalian 116030, China § School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116023, China ∥ Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China S Supporting Information *

ABSTRACT: This article describes the use of surface plasmon resonance (SPR) spectroscopy to study antibody−ligand interactions for hydrophobic charge-induction chromatography (HCIC) and its versatility in investigating the surface and solution factors affecting the interactions. Two density model surfaces presenting the HCIC ligand (mercapto-ethyl-pyridine, MEP) were prepared on Au using a self-assembly technique. The surface chemistry and structure, ionization, and protein binding of such model surfaces were characterized by X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure (NEXAFS), contact-angle titration, and SPR, respectively. The influences of the surface and solution factors, e.g., ligand density, salt concentration, and solution pH, on protein adsorption were determined by SPR. Our results showed that ligand density affects both equilibrium and dynamic aspects of the interactions. Specifically, a dense ligand leads to an increase in binding strength, rapid adsorption, slow desorption, and low specificity. In addition, both hydrophobic interactions and hydrogen bonding contribute significantly to the protein adsorption at neutral pH, while the electrostatic repulsion is overwhelmed under acidic conditions. The hydrophobic interaction at a high concentration of lyotropic salt would cause drastic conformational changes in the adsorbed protein. Combined with the selfassembly technique, SPR proves to be a powerful tool for studying the interactions between an antibody and a chromatographic ligand.

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INTRODUCTION The growing demand for antibodies in therapeutic and diagnostic applications calls for industrial-scale purifications with low cost, high efficiency, and high purity.1−4 Recently, hydrophobic charge-induction chromatography (HCIC) has become a promising technique for antibody purification.5−7 The molecular basis of HCIC is the interactions between antibodies and ionizable ligands, e.g., mercapto-ethyl-pyridine (MEP).8,9 The nature of such interactions can be modulated by pH. Neutral pH favors hydrophobic interactions, and acidic conditions promote electrostatic repulsion. Accordingly, protein adsorption is caused by the hydrophobic interaction between protein and the uncharged ligand at neutral pH, and the desorption process takes place due to the electrostatic repulsion between the charged ligand and protein under acidic conditions (Scheme 1). Molecular dynamic results revealed that the headgroup of the HCIC ligand, i.e., pyridine in the MEP molecule, primarily prefers to interact with some of the hydrophobic pockets on the Fc fragment of IgG.10 Such specificity toward the Fc fragment has allowed successfully applications of HCIC to purify antibodies and Fc-fusion proteins from a variety of feedstocks (e.g., serum, ascites, and culture media).11,12 Despite its excellent performance in © 2015 American Chemical Society

Scheme 1. (A) Antibody Adsorption on Hydrophobic Charge Induction Chromatography (HCIC) and (B) the Mercapto-ethyl-pyridine (MEP) Ligand for pH-Dependent Dual-Mode Interactions in HCIC

practice, it is desirable to understand the protein−ligand interactions for the elucidation of the underlying molecular mechanism. Received: November 18, 2014 Revised: February 25, 2015 Published: March 3, 2015 3422

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(5 mM in ethanol) for 1 h at 25 °C to form a hydroxyl-terminated selfassembled monolayer (OH surface). The resulting surfaces were cleaned in a stirring ethanol bath. The cleaned surfaces were dried under a stream of nitrogen and then immersed in 10% DVS solution (v/v, 0.5 M carbonate buffer, pH 11) for 1 h at 25 °C. The DVStreated samples (OH-DVS surface) were thoroughly rinsed with water and ethanol, dried with a stream of nitrogen, and stored in the dark at 4 °C. DVS-MEP Surface. The DVS-treated samples were immersed in MEP solution (50 mM in carbonate buffer (0.5 M, pH 11) containing 10% v/v acetone) at 25 °C for 1 h to obtain the DVS-MEP surfaces. The surfaces were then thoroughly rinsed with water and ethanol, dried under a stream of nitrogen, and stored in the dark at 4°C. For the SPR study, the DVS-MEP surfaces were immersed in oligo(ethylene glycol) thiol solution (5 mM in carbonate buffer (10 mM, pH 8)) for 1 h to deactivate any remaining vinyl sulfone groups and then rinsed with water, dried under a stream of nitrogen, and stored at 4 °C. MEP-Only Surface. Fresh Au substrates were immersed in MEP solution (50 mM in carbonate buffer (0.5 M, pH 11) containing 10% v/v acetone) at 25 °C for 1 h to form self-assembled monolayers (MEP-only surface). The resulting surfaces were thoroughly rinsed with water and ethanol, dried with a stream of nitrogen, and stored in the dark at 4 °C. Contact Angle Measurement. Water contact angles on the two types of MEP surfaces, i.e., DVS-MEP and MEP-only surfaces, were measured using a contact angle goniometer (DH-HV1351UM, Beijing, China). The measurement of contact-angle titration has been reported elsewhere.21 At least three spots on two or more replicates of each sample were analyzed. The reported values are averaged over the multiple spots and presented with standard deviations. X-ray Photoelectron Spectroscopy (XPS). A Thermo Scientific ESCALAB 250Xi X-ray photoelectron spectrometer using monochromatic Al Kα radiation was utilized to characterize the DVS-MEP and MEP-only surfaces at a takeoff angle of 90° from the surface. All binding energies were referenced to the C 1s peak calibrated at 285.0 eV. Two spots on two replicates of each sample for composition were analyzed. The reported values are averaged over the multiple spots and presented with standard deviations. Near-Edge X-ray Absorption Fine Structure Spectroscopy. Near-edge X-ray absorption fine structure (NEXAFS) spectroscopy probes the molecular structure of surface-adsorbed species by measuring characteristic absorption resonances corresponding to electronic transitions from atomic core levels to unoccupied molecular orbitals. 22 NEXAFS spectra were collected at the National Synchrotron Light Source (NSLS) U7A beamline at Brookhaven National Laboratory (Upton, New York) using an elliptically polarized beam with ∼85% p polarization. This beamline is equipped with a monochromator (600 lines/mm grating) which provides a full width at half-maximum resolution of ∼0.15 eV at the carbon K edge. At the carbon K edge, the monochromator energy scale was calibrated using the intense C 1s−π* transition at 285.35 eV of a graphite transmission grid placed in the path of the X-rays, and the partial electron yield was monitored by a detector with the bias voltage maintained at −150 V. Samples were mounted to allow rotation and changes to the angle between the sample surface and the synchrotron X-rays. The NEXAFS angle is defined as the angle between the incident light and the sample surface. SPR Spectroscopy. A Biosuplar-400T SPR spectrometer (Analytical μ-Systems, Germany) with a light-emitting diode light source (λ = 670 nm), high-refractive-index prism (n = 1.61), and two-channel flow cell was used for all SPR measurements. The flow rate was set at 50 μL/min. For protein adsorption measurements, the following protocol was used: a buffered protein solution was allowed to flow for 10−30 min, followed by a buffer washing step for 10 min, a regeneration step with 0.01 M NaOH for 2 min, and a final buffer washing step for 5 min. The running buffer used in all the SPR experiments was 20 mM phosphate buffer (pH 7) unless otherwise stated.

Surface plasmon resonance (SPR) is a powerful tool for examining protein−ligand interactions.13,14 By monitoring refractive index changes close to the sensor surface, SPR is capable of detecting the interactions between protein and the sensor surface in real time. With the aid of self-assembly and subsequent surface modification, ligands of interest can be presented on the sensor surface in a controlled manner. Once the protein and buffer solutions are sequentially pumped over the sensor surface, the time-resolved adsorption and desorption process is obtained. The equilibrium and dynamic aspects of the interactions between the protein and ligand of interest, e.g., binding strength and adsorption and desorption kinetic rate constants, can be calculated. The obtained information allows one to evaluate chromatographic ligands quantitatively.15−17 By controlling the composition of the solution and selecting target proteins, e.g., salt chaotropicity and protein hydrophobicity, the fundamental physics underlying the interactions can be elucidated.18 In this work, model surfaces presenting two different densities of MEP ligands are prepared using self-assembly and subsequent surface modification. The two MEP surfaces were characterized by X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure (NEXAFS), contactangle titration, and SPR protein binding experiments. The influences of the surface and solution factors, i.e., ligand density, salt concentration, and solution pH, on the interactions between the antibody and MEP ligand are examined. Our SPR results established the use of SPR to study antibody− ligand interactions for HCIC and its versatility in quantitatively investigating the effects of various surface and solution factors on such interactions.

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EXPERIMENTAL SECTION

Reagents and Materials. 11-Mercaptoundecanol and bovine serum albumin (BSA) were obtained from Sigma (Milwaukee, WI, USA). Bovine serum γ-globulin (IgG, electrophoresis purity 99.0%) was obtained from Merck KGaA (Darmstadt, Germany). 2-Mercapto1-methylimidazole (MMI) was purchased from Aladdin (Shanghai, China). Human γ-globulin from human serum (hIgG 1, purity >95%) was obtained from Wako Pure Chemical Industries (Osaka, Japan). Intravenous human immunoglobulin (hIgG 2, ∼50 mg/mL) was obtained from Sichuan Yuanda Shuyang Pharmaceutical (Chengdu, China). HyClone newborn calf serum (0.1 μm sterile filtered) was purchased from Thermoscientific (Tauranga, New Zealand). Oligo(ethylene glycol) thiol was synthesized as previously described.19 Mercapto-ethyl-pyridine (MEP) was provided by Prof. J. Ren at Dalian University of Technology. Ethanol (anhydrous, Spectro) was purchased from Tedia (Fairfield, OH, USA). Other chemicals were of analytical grade from local sources. All chemicals were used as received without further purification. Silicon wafers were purchased from Tianjin Semiconductor Technology Institute (Tianjin, China). Ultrapure Millipore deionized water with an 18.2 MΩ·cm resistivity was used in all experiments. Unfunctionalized bare gold chips for SPR were purchased from Analytical μ-Systems (Regensburg, Germany). Preparation of Au Substrates. Silicon wafers were immersed in piranha solution (a 3:1 mixture of 97% sulfuric acid and 30% hydrogen peroxide) for 30 min. (Caution! Piranha solution reacts violently with most organic materials and must be handled with extreme care.) Once the wafers were removed from piranha solution, they were rinsed with copious amounts of water and ethanol and then dried under a stream of nitrogen. Titanium (5 nm) and gold (45 nm) were sequentially deposited onto the cleaned silicon wafers (10 mm × 10 mm) using a Turbo Sputter Coater K575XD (Kent, U.K.). DVS-Treated Surface. The divinyl sulfone (DVS) treatment of hydroxyl-terminated surfaces has been reported elsewhere.20 Briefly, fresh Au substrates were immersed in 11-mercaptoundecanol solution 3423

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Langmuir Adsorption Equilibrium and Kinetics by SPR. Buffers containing different concentrations of proteins (0.01−3.0 mg/mL) flowed over the sensor surfaces for at least 30 min. After the adsorption reached equilibrium, the surfaces were regenerated and the equilibrium protein concentrations were determined at 280 nm with the BioSpectrometer (Eppendorf, Germany). The adsorption isotherm was fitted to the Langmuir equation

R eq =

by an order of magnitude. Method A is a stepwise process to immobilize MEP ligands via DVS-based chemistry.20 A hydroxyl-terminated self-assembled monolayer (OH surface) on Au as the starting surface was treated with DVS, followed by incubation with MEP ligands under alkaline conditions (pH 11) to fabricate the DVS-MEP surface. This two-step method has also been demonstrated by Ratner and coworkers to successfully to functionalize hydroxyl-terminated surfaces with thiolate glycans.20,28,29 The stepwise process of DVS-based chemistry on SAMs was confirmed using XPS and time-of-flight secondary ion mass spectrometry (ToF-SIMS).20 The reported density of the thiolate ligands in that study was roughly estimated to be 0.3 molecule/nm2.20 Method B is a direct selfassembly of MEP ligands on bare Au to form an MEP-only surface, which presents MEP ligands densely. As model surfaces presenting aromatic groups, the pyridine-terminated SAM surfaces have been well characterized using NEXAFS,30 IR,31 XPS,32 and electrochemistry.33 With the aid of in situ scanning tunneling microscopy,34,35 the density of pyridine groups presented on highly packed and defect-free SAMs was reported to be close to 3 molecules/nm2. Characterization of MEP Surfaces by XPS and NEXAFS. To examine the effectiveness of DVS-based chemistry and the reproducibility of the MEP surfaces prepared in our study, each surface-modification step and subsequent treatment was characterized by XPS. The XPS survey scans and the corresponding organic compositions are shown in Figure 1a and Figure S1, respectively. The survey scans in Figure 1a display the specific lines of Au (Au 4f, 4d, and 4p) as the main features and carbon (C 1s) as well. The detailed scans of nitrogen confirmed nitrogen species on both MEP surfaces, originating from the pyridine ring in the MEP molecule. The detailed scans of oxygen established the existence of oxygen species on the OH, OH-DVS, and DVS-MEP surfaces, attributed to hydroxyl groups and sulfone groups. However, an unexpected oxygen peak is observed on the MEP-only surface. This discrepancy is likely due to tightly bound water molecules with nitrogen species in the pyridine ring.36 Although the theoretical ratio of oxygen to sulfur for OH SAMs is 1.0, the experimental ratio determined by XPS (1.7 ± 0.2) is higher than 1. This discrepancy can be explained by the sulfur signal attenuation by the monolayers. Compared to the OH SAMs, slight changes in the O/S ratio (1.8 ± 0.2 for the

R maxCeq Kd + Ceq

(1)

where Req and Ceq are the SPR response to equilibrium adsorption and the equilibrium protein concentration, respectively. Rmax is the maximum binding capacity, and Kd is the dissociation constant.

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RESULTS AND DISCUSSION Preparation of MEP Surfaces. Ligand density has been identified to be a key factor greatly affecting antibody adsorption on HCIC adsorbents.23−25 Using the self-assembly technique, controlled ligand density can be conveniently realized on SPR sensor chips.26,27 Here, we used two methods (illustrated in Scheme 2) to present MEP densities that differ Scheme 2. Two Methods of Preparing Surfaces Presenting MEP Ligandsa

a

Method A: DVS-MEP surface via a stepwise modification.20 Method B: MEP-only surface via direct self-assembly.

Figure 1. XPS spectra of (a) survey scans, (b) sulfur 2p, and (c) carbon 1s for (1) OH, (2) OH-DVS, (3) DVS-MEP, and (4) MEP-only surfaces. 3424

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Langmuir OH-DVS surface and 1.6 ± 0.1 for the DVS-MEP surface) could be attributed to sulfone groups in the DVS molecule and the mercapto group in the MEP molecule. The O/N ratio was calculated to be 14.0 ± 0.5 for the DVS-MEP surface. Taking account of highly packed and defect-free alkanethiol SAMs with a density of 4.67 molecule/nm2,37 the estimated density for the MEP ligand on the MEP-DVS surface is roughly 0.3 molecule/ nm2. To establish that DVS functionality is introduced onto the OH surface, XPS sulfur 2p peaks for each surface are analyzed (Figure 1b). On the OH and MEP-only surfaces, both S 2p spectra exhibited a typical S 2p doublet at around 162 eV, corresponding to thiolate species bonding to Au substrates.38 On both OH-DVS and DVS-MEP surfaces, however, an extra doublet around 168 eV was observed, which is commonly assigned to the oxidized sulfur species. A control OH surface incubated in basic buffer without subsequent treatment was also analyzed. The absence of an oxidized sulfur signal on the control surface excluded the fact that the oxidized sulfur species originate from the oxidation of the thiolate species during sample handling. These results indicate that the observed oxidized sulfur species at 168 eV can be attributed to sulfone groups.39 To confirm that the DVS treatment does not deteriorate the structure of SAMs, XPS carbon 1s peaks for each surface were examined (Figure 1c). The C 1s spectrum of the OH surface showed two peaks following peak assignment: a main peak at 285.0 eV that is characteristic of the repeat units of the tail methylene chain and another peak at around 286.5 eV that corresponds to carbon atoms adjacent to the hydroxyl groups. After treatment with DVS, an additional peak at around 286.0 eV was observed, which most likely corresponds to the carbon atoms adjacent to the sulfone groups. When the OH-DVS surface was further treated with MEP, the intensity of the 286 eV peak increased, likely due to the pyridine ring in the bound MEP. To confirm this peak assignment, a control sample of the MEP-only surface was analyzed (Figure 1c). As we can see, a small peak at around 286.0 eV corresponding to the ortho and para carbon atoms of the pyridine ring was detected, consistent with the reported value for pyridine-terminated SAMs.30 To further support the fact that the DVS treatment does not deteriorate the structure of SAMs, the ordering of SAMs before and after DVS treatment (OH and OH-DVS surface) was examined by NEXAFS. Carbon K-edge spectra acquired at 90 and 20° are presented in Figure S2. In the spectra collected from both surfaces, we observed a small pre-edge feature representative of π* CC orbitals.40 For the OH surface, this peak is most likely due to hydrocarbon contamination on the surface, but for the OH-DVS surface, this peak can also correspond to the CC bond of the terminal vinyl sulfone groups immobilized on the monolayer. At higher X-ray energies, both sets of samples exhibited a shoulder at 288 eV and a broad resonance at 293 eV, related to R*/C−H σ* and C−C σ* molecular orbitals, respectively.36,41 The orientation and ordering of molecular bonds can be determined by simply following any change in the X-ray absorption as we rotate the sample and vary the incident angle of the X-rays. Difference spectra (90−20°) collected from the OH and OH-DVS surfaces can be found in Figure S2. The two difference spectra demonstrate a high degree of ordering of both the R*/C−H σ* (large positive dichroism) and C−C σ* molecular orbitals (large negative dichroism). These identical difference spectra

suggest that the DVS treatment does not disrupt the order of the SAM hydrocarbon chains. Altogether, the results obtained from XPS and NEXAFS imply that (1) vinyl sulfone groups are introduced onto the OH surface; (2) the SAMs structure is preserved after DVS treatment; (3) the MEP density on the DVS-MEP surface is ∼0.3 molecule/nm2; and (4) each surface modification step and subsequent treatment (illustrated in Scheme 2) is effective and reproducible. Characterization of MEP Surfaces by Contact Angle Titration and SPR Protein Binding. Contact angle measurements were carried out to track the stepwise preparation of the DVS-MEP surface. The starting surface (OH surface) has a water contact angle of 44.3 ± 0.6°. After DVS treatment, the contact angle increases to 65.8 ± 0.3°, which reflects the surface changing from polar hydroxyl groups to less-polar vinyl sulfone groups. The final DVS-MEP surface has a water contact angle of 59.0 ± 0.8° due to the pyridine ring of the MEP ligand being more polar than the vinyl sulfone group. For the MEP-only surface, a comparable level of contact angle (58.7 ± 0.5°) was observed. To probe the ionization of the surface-immobilized MEP groups, we examined contact angles of water on the two types of MEP surfaces after treatment with a series of buffer solutions as a function of pH. The contact-angle titrations of the two MEP surfaces are shown in Figure 2. As can be seen, the

Figure 2. Representative photomicrographs of water droplets on (a) DVS-MEP and MEP-only surfaces treated with different pH buffers and (b) contact-angle titrations for DVS-MEP (■) and MEP-only (●) surfaces.

surfaces treated with acidic buffers are more hydrophilic than those treated with neutral and basic buffers. This is because the acidic aqueous solutions ionize the tertiary amine in the pyridine ring of the MEP groups on the surfaces, which in turn decrease the water contact angles. For both MEP surfaces, a transition in contact angle is observed between pH 3 and pH 5. It is noteworthy that, regardless of the type of MEP surfaces, the transition pH is slightly lower than the reported pKa value (4.8) of MEP molecules in solution. Similar acidic shifts in pKa were also reported on amine-terminated surfaces.42 For example, the pKa value of NH2-terminated SAMs is 6.5, whereas the pKa of the corresponding NH2-containing selfassembled molecule is 8.9 in the bulk solution. The contactangle titration study proved that both the DVS-MEP and the 3425

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observed similar binding profiles of the two human IgG proteins, which suggests that our methods are applicable to various immunoglobulin proteins. Influence of Ligand Density on the Interactions. By varying the MEP ligand density, equilibrium and dynamic aspects of the antibody−ligand interactions, namely, the binding strength, and adsorption and desorption kinetic rate constants, were compared. The study of BSA is included because albumin is the most common impurity coadsorbed with IgG in practice.9 The adsorption isotherms of IgG and BSA on the DVS-MEP and MEP-only surfaces are shown in Figure 4. The isotherm data were fitted to a Langmuir model

MEP-only surfaces present MEP ligands, which can be ionized under acidic conditions. SPR was used to examine protein interactions with the surface-immobilized MEP groups. The time-resolved protein adsorption and desorption process was obtained. As shown in Figure 3a, the DVS-MEP surface shows a significant increase in

Figure 3. Typical SPR sensorgrams of protein interactions with the DVS-MEP surface. (a) IgG adsorption, desorption, and surface regeneration and (b) examples of 50 continuous binding−regeneration cycles on the DVS-MEP surface. Aqueous solutions pumped over the DVS-MEP surface are (1) pH 7 buffer, (2) IgG, and (3) 0.01 M NaOH. A dip (indicated by *) is due to the change in the bulk refractive index.

Figure 4. Adsorption isotherms for IgG (■) and BSA (●) adsorption on (a) DVS-MEP and (b) MEP-only surfaces with Langmuir fitting (solid lines).

SPR response due to protein adsorption as IgG solution flowed over the surface. Once the solution was switched to buffer, weakly bound protein started to desorb. The efficient desorption of bound protein occurred by reducing the buffer pH. As acidic buffer solution (pH 3) was passed over the bound protein film, and 80% or more bound proteins were removed (data not shown). This finding is consistent with the fact that the adsorbed IgG can be removed by electrostatic repulsion under acidic conditions.7,8 The surface can be regenerated using either a basic solution (0.01 M NaOH) or an arginine solution (0.01 M), and most residual proteins were washed off. Owing to the intrinsic properties of SAMs on Au, the surfaces presenting MEP ligand are very stable and reusable for SPR experiments (Figure 3b). The surface retained 95% of its binding capacity after 50 continuous binding−regeneration cycles on SPR. To obtain a better understanding of the ligand−protein interactions on the MEP surfaces, a set of protein binding experiments on the OH and OH-DVS surfaces were carried out (Figure S3). As we can see, small amounts of IgG and BSA adsorbed to the OH surface, consistent with the fact that OHterminated surfaces are low protein fouling.43 On the OH-DVS surface, however, remarkable amounts of adsorbed proteins were irreversibly bound, which means that vinyl sulfone groups, introduced onto the OH surface, are chemically reactive to thiol and amine species.20 These results also support our early findings obtained by XPS. To establish that our model surfaces are applicable to the study of immunoglobulin proteins from different hosts, two types of human IgGs, i.e., hIgG 1 and hIgG 2, were used in protein binding experiments (Figure S4). Compared to IgG obtained from bovine (Figure 3a), we

(eq 1) to yield the dissociation constant (Kd) and the maximum binding capacity (Rmax). The calculated values of Kd and Rmax are summarized in Table S1. For IgG binding, the DVS-MEP surface results in a Kd of 1.48 × 10−6 M, whereas the MEP-only surface yields a Kd of 1.16 × 10−7 M. For BSA binding, the DVS-MEP surface results in a Kd of 1.16 × 10−5 M, and the MEP-only surface yields a Kd of 1.42 × 10−5 M. Thus, the MEP-only surface shows an order of magnitude higher affinity toward IgG and the same level of affinity toward BSA when compared to the DVS-MEP surface. Although the high affinity of the MEP-only surface/IgG is the same as for protein A/IgG (1.4 × 10−7 M),16 the specificity of this surface is surprisingly low. Note that the specificity is assessed by the ratio of the maximum IgG binding to the maximum BSA binding.16,44 The calculated ratio for the MEPonly surface is 1.2:1, whereas the value for DVS-MEP is 7.8:1. The low specificity of the MEP-only surface was also supported by the serum adsorption experiments (Figure S5). When diluted serum (0.1 and 1%) flowed over the two MEP surfaces, the amount of serum adsorbed on the MEP-only surface was higher than that adsorbed on the DVS-MEP surface. This difference in specificity could be explained by the immobilized MEP density being higher on the MEP-only surface than on the DVS-MEP surface. Typically, HCIC adsorbents exhibit densitydependent specificity for target antibody proteins.23,45 For example, 2-mercapto-1-methylimidazole (MMI), a new HCIC ligand, also showed reduced specificity at high surface density (Table S1). 3426

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adsorption. In the absence of salt, both hydrogen bonding and the hydrophobic interaction would contribute to IgG adsorption. As the salt concentration increases, the extent of IgG adsorption decreases first, suggesting that the added salt weakens the hydrogen bonding.47,48 At an even higher salt concentration, the hydrophobic interaction would be dominant because of the salting-out effect,49 in which the bound water molecules on both protein and ligand surfaces are removed. Accordingly, more proteins adsorbed on the two MEP surfaces. In addition, the degree of the influence is dependent on the ligand density. The presence of salt was able to reduce nearly 90% of IgG adsorption on the DVS-MEP surface, whereas the maximum reduction in IgG adsorption on the MEP-only surface by the addition of salt was around 40%. We believe that the tolerance of salt in the case of a dense ligand surface is probably due to the overwhelmed hydrophobic interaction. The higher salt concentrations would also affect the IgG adsorption process (Figure 5b). Mass overshoots (indicated by the arrows in Figure 5b) were observed in the SPR sensorgrams. These mass overshoots suggest that the adsorbed proteins undergo conformational changes or reorientation. Similar mass overshoots observed by SPR have been reported ́ group50 on various well-defined surfaces and by Garcia’s Whitesides’ group51 on CH3-terminated surfaces. Typically, an increase in surface hydrophobicity causes an increase in the degree of structural change during the adsorption process.52 Besides SPR, a variety of spectroscopic methods, e.g., circular dichroism,52 total internal reflectance fluorescence,53 ToFSIMS, and sum frequency generation,54 have been developed to investigate protein unfolding induced by adsorption. A few HCIC mechanistic studies have investigated the protein conformational changes or reorientation during adsorption to HCIC ligand-bearing surfaces.25 Furthermore, MD simulation on protein−HCIC ligand interaction provided molecular insights into the dynamic process of protein adsorption55,56 and quantitatively compared the conformational changes affected by salt type and concentration.25 It revealed that the high concentration of lyotropic salt promotes hydrophobic interaction, which in turn causes significant unfolding of the adsorbed protein. Our observation by SPR provides experimental support for the simulation results, suggesting that the hydrophobic interaction would contribute to protein adsorption onto HCIC ligands. Influence of Solution pH on the Interactions. On the two MEP surfaces, the extent of protein adsorption was examined by varying the pH values of the protein solutions. Figure 6 illustrates the influence of pH on IgG and BSA adsorption on the two MEP surfaces as measured by SPR. As shown in Figure 6, a large amount of IgG adsorption on the DVS-MEP surface is observed at the range of pH 5−8.5, and the maximum is reached at pH 6. As the pH decreases to 4, the amount of IgG adsorption decreases remarkably. The reported values of pI for IgG and pKa for MEP are approximately 6.0− 8.5 and 4.8, respectively. By lowering the solution pH, strong electrostatic repulsion between IgG and MEP surfaces appears to overcome the hydrophobic interaction and hydrogen bonding, resulting in a significant reduction in IgG adsorption. For BSA, under all tested pH values with the exception of pH 4, its adsorbed amount is much lower than that from the IgG study, and the maximum adsorption is reached at pH 5. Such different adsorption behaviors in response to the change in pH allow one to utilize the solution pH to tune the adsorption specificity. For example, the ratio of adsorbed IgG to adsorbed

Besides the dissociation constant (Kd), adsorption and desorption kinetic rate constants (ka and kd, respectively) describe the dynamic aspect of the protein−ligand interactions. The values of ka and kd were obtained by fitting the timeresolved SPR response of IgG adsorption to eqs S1 and S2 as summarized in Table 1. For the two MEP surfaces, the best fits Table 1. Summary of ka and kd for IgG on the DVS-MEP and MEP-Only Surfaces ka (103 M−1 s−1) kd (10−3 s−1)

DVS-MEP

MEP-only

1.95 2.89

7.50 0.87

were achieved for IgG concentrations closed to or lower than their Kd values (Figure S6). Compared to the DVS-MEP surface, the MEP-only surface exhibits a rapid adsorption process and a slow desorption process. A number of factors, such as the ligand density, ligand structure, pH, solution composition, and resin pore size, have been identified to affect the protein−ligand interactions.7,24,45 Among them, ligand density affects protein adsorption greatly. Although the density effects have been investigated intensively by macroscopic methods and molecular dynamics (MD) simulation, its effects on the dynamic aspects of the interactions have not been examined due to the lack of experimental tools.46 Our study demonstrates that SPR is powerful in quantifying both the dynamic and equilibrium aspects at the interface. In particular, our results show that the increase in ligand density results in an increase in the adsorption rate and a decrease in the desorption rate, leading to enhanced interaction strength. Influence of Salt Concentration on the Interactions. On the two MEP surfaces, the extent of IgG adsorption was examined at various salt concentrations under a neutral condition. The extents of IgG adsorption on the DVS-MEP and MEP-only surfaces are plotted in Figure 5a. As can be seen, the U-shaped curves of IgG adsorption appear with the increase in salt concentration. This tendency is due to the hydrophobic interaction combined with hydrogen bonding during IgG

Figure 5. Effect of Na2SO4 concentration on (a) the extent of IgG adsorption on DVS-MEP (■) and MEP-only (●) surfaces and (b) the typical SPR sensorgrams of IgG adsorption on the MEP-only surface. The SPR responses were normalized to the maximum IgG adsorption on each set of the MEP surfaces. 3427

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experimental ratios of IgG to BSA on surfaces. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: ff[email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Joe E. Baio (Oregon State University) and Tobias Weidner (Max Planck Institute for Polymer Research) for NEXAFS data acquisition and Daniel Fischer (NIST) for providing experimental equipment for NEXAFS spectroscopy and technical assistance at the synchrotron. NEXAFS studies were performed at the NSLS, Brookhaven National Laboratory, which is supported by the U.S. Department of Energy, Division of Materials Science and Division of Chemical Sciences. This work was supported by the Natural Science Foundation of China (Nos. 21104008, 21231003, and 21276228), the “111” project of the Ministry of Education of China, and the Fundamental Research Funds for the Central Universities (DUT13LAB07).

Figure 6. pH effect on the adsorption of IgG (black bar) and BSA (white bar) on (a) DVS-MEP and (b) MEP-only surfaces. The SPR responses were normalized to the maximum IgG adsorption on each set of MEP surfaces.

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BSA on the DVS-MEP surface is 9.3 at pH 5, whereas this ratio is increased to 17.9 at pH 6, implying a much higher adsorption capacity toward IgG with a simple change in pH. The experimental ratios of IgG to BSA on the DVS-MEP and MEP-only surfaces in different pH buffers are summarized in Table S2. This information can be used to select the proper solution pH for the practical purification of IgG from BSAcontaining feedstocks.

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CONCLUSIONS This article demonstrates the use of SPR to study antibody− ligand interactions for HCIC and its versatility in investigating the surface and solution factors affecting the interactions. By comparing model surfaces presenting two different MEP densities, we revealed that both equilibrium and dynamic aspects of the interactions are affected by the ligand density greatly. In particular, dense MEP ligands lead to increase in binding strength, rapid protein adsorption, slow protein desorption and low specificity. By varying solution compositions, e.g., salt concentration and pH, our SPR results suggest that both hydrophobic interactions and hydrogen bonding between protein and uncharged ligand are present at neutral pH, and the hydrophobic interaction at high concentrations of lyotropic salt may cause the conformational changes or reorientation of the adsorbed proteins. We envision that SPR combined with the self-assembly technique will be useful for elucidating the fundamentals underlying the protein−ligand interactions on the surfaces, screening new chromatographic ligands and selecting chromatographic adsorbents with appropriate ligand density and operation conditions. As a new tool to study HCIC, more work should be focused on a series of ligand densities to establish a general correlation between the interactions and the surface density.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

XPS analysis of composition comparison, NEXAFS analysis of SAM ordering, SPR sensorgrams of proteins binding on surfaces, Kd and Rmax of protein on surfaces, SPR experimental and theoretical fitting of protein binding on surfaces, and 3428

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