Binary Adsorption Processes of Albumin and Immunoglobulin on

Article pubs.acs.org/jced

Binary Adsorption Processes of Albumin and Immunoglobulin on Hydrophobic Charge-Induction Resins Qilei Zhang,† Ferdinand Schimpf,†,‡ Hui-Li Lu,† Dong-Qiang Lin,*,† and Shan-Jing Yao† †

Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China ‡ Institute of Biochemical Engineering, Technische Universität München, 85748 Garching, Germany ABSTRACT: Hydrophobic charge-induction chromatography with 4mercaptoethyl-pyridine as ligands shows promising application in antibody purification. In this study, competitive adsorption of protein mixtures composed with bovine serum albumin (BSA) and immunoglobulin (IgG) was investigated with MEP HyperCel. Static adsorption and dynamic binding processes were measured under different media pH and BSA/IgG mass ratios. The results showed that MEP HyperCel had high pH-dependent selectivity. BSA can be adsorbed quicker than IgG but part of the adsorbed BSA would be gradually displaced by IgG as a result of competitive adsorption. The effects of NaCl and (NH4)2SO4 on protein mixture adsorption showed that both salts can enhance IgG selectivity on MEP HyperCel, but the effect was different based on the combination of electrostatic and hydrophobic interactions. Competitive adsorption mechanism was discussed and the results obtained would be useful in the separation of albumin and immunoglobulin from protein mixtures.

1. INTRODUCTION

However, protein adsorption selectivity of MEP HyperCel still needs to be improved as compared to Protein A affinity chromatography. For example, albumin widely exists in blood serum and cell culture media, and the presence of albumin can significantly affect the adsorption process of immunoglobulin G (IgG) with HCIC ligands.11,12 The effects of impurities or other proteins on target protein purification have been studied with various techniques and different resins. Cramer et al.13 investigated a selective desorption process on ceramic hydroxyapatite for the purification of monomeric antibody from associated aggregates and post-Protein A impurities. A 100% yield of pure monomeric antibody was achieved after mobile phase optimization for selective desorption. Carta and Lewus14 developed an approximate rate equation to describe the kinetics of multicomponent adsorption, which can be used in the numerical simulation of adsorption systems with concentration-dependent micropores. Martin et al.15 theoretically analyzed multicomponent adsorption kinetics for protein adsorption in porous ion exchangers, and they found the experimental results agreed well with simulation. Confocal laser scanning microscopy (CLSM) is a powerful technique for visualizing protein distribution profiles in porous chromatographic resins, which is useful in studying competitive adsorption processes of different proteins in resins. Shi et

Antibodies with important biological functions have shown wide applications in diagnostic and therapeutic treatments.1 Currently, antibody drugs are one of the largest drug categories in the pharmaceutical market and there are new antibody drugs being approved by government bodies.2 With the development of cell expression techniques, downstream processing such as protein purification have become the major contributor in the total cost of antibody production.3,4 Protein A-based affinity chromatography is a standard technique in large-scale antibody purification owing to its excellent selectivity.5 However, some well-known limitations of Protein A, such as high cost, low reusability, and ligand leaching, force the industry to explore new resins and processes.6 Hydrophobic charge-induction chromatography (HCIC) is a promising technique that has been successfully applied in antibody purification.7,8 It is considered to be superficially similar to Protein A chromatography that involves hydrophobic interactions for adsorption and elution because of ionization of histidine residues.9 HCIC can capture antibodies under physiological conditions via hydrophobic interactions and achieve effective elution through electrostatic repulsion. 4Mercaptoethyl-pyridine (MEP) is a typical HCIC ligand with a pKa value of 4.8, and a resin using MEP as the ligand has been developed which is the first commercialized HCIC resin MEP HyperCel. It has been applied to purify monoclonal antibodies from different resources and showed high dynamic binding capacities.10 © 2016 American Chemical Society

Received: December 31, 2015 Accepted: February 23, 2016 Published: February 29, 2016 1353

DOI: 10.1021/acs.jced.5b01108 J. Chem. Eng. Data 2016, 61, 1353−1360

Journal of Chemical & Engineering Data

Article

al.16 used CLSM to study dynamic adsorption behaviors of two intrinsic fluorescent proteins in the Q Sepharose HP resin. The fluorescent images showed that the two proteins exhibited distinct fading rates as compared to single component studies. EI-Sayed and Chase17 also used CLSM to study competitive adsorption of α-lactalbumin and β-lactoglobulin on SP Sepharose FF, and the results showed that the protein distribution in resins were different between a singlecomponent system and a two-component system, and βlactoglobulin was displaced by α-lactalbumin despite the lower affinity of α-lactalbumin under the experimental conditions. One of the problems of CLSM is it usually requires protein labeling with a fluorescent probe, which may affect the affinity of proteins on adsorbents.18 To make HCIC applicable for the separation of a variety of protein candidates, traditional adsorption isotherms and kinetic studies are still needed to help understand adsorption processes, and more data should be collected and analyzed to understand adsorption processes and mechanisms. In this study, Human IgG and bovine serum albumin (BSA) were used as the model target and impurity, and dynamic binding processes of IgG/BSA mixtures on MEP HyperCel were investigated under different media pH and protein mixture mass ratios. The effect of salts on the competitive adsorption process was also studied and the mechanism of competitive adsorption was discussed.

2.4. Protein Concentration Analysis. HPLC analysis was used to measure protein concentrations in liquids. It was performed with an LC-3000 HPLC system (Beijing Chuangxintongheng Science & Technology Co., Ltd., Beijing, China) using a TSK G3000SWXL column (7.8 mm × 30.0 mm, Tosoh Bioscience, Tokyo, Japan). The mobile phase was 0.1 M Na2SO4 in 0.1 M phosphate solution (pH 6.7). A sample injection volume of 20 μL with a mobile phase flow rate of 0.5 mL/min was applied, and the detection wavelength was 280 nm. All samples were filtered through a 0.22 μm microporous membrane in advance.

3. RESULTS AND DISCUSSION 3.1. Static Adsorption. MEP HyperCel is a typical HCIC resin and the protein adsorption is usually pH-dependent. Figure 1 shows the saturated adsorption capacities (Qm) of

2. MATERIALS AND METHODS 2.1. Materials. MEP HyperCel was purchased from Pall Life Sciences (NY, USA). Bovine serum albumin (BSA) was bought from Sigma (Milwaukee, USA). Human IgG was obtained from Wako Pure Chemical Industries (Wako, Japan). All other chemicals were of analytical grade. 2.2. Adsorption Isotherms. Static adsorption of IgG and BSA on MEP HyperCel was studied by batch adsorption equilibrium experiments. Citrate phosphate buffers with pH of 5−8 were used as the experimental media. For the experiments, 0.8 mL of protein solutions (single-component, or protein mixtures with various BSA/IgG mass ratios) with protein concentration ranging from 2−40 mg/mL was mixed with about 40 mg of drained MEP HyperCel resin. The mixture was then shaken in a thermoshaker at 25 °C for 3 h to achieve adsorption equilibrium. The samples were then filtered with 0.22 μm membrane filtration and analyzed by high performance liquid chromatography (HPLC). The amount of adsorbed protein was calculated, and Langmuir adsorption eq (eq 1) was used for data analysis. Q* =

Figure 1. Saturated adsorption capacities (Qm) of individual BSA (white) and IgG (black) on MEP HyperCel under different pH.

individual BSA and IgG with MEP HyperCel under a pH of 5.0−8.0. The results show that IgG had the Qm above 160 mg/ mL when pH was around 6−8, while BSA showed a maximum Qm of 65 mg/mL at pH 5, and the adsorption capacity decreased dramatically with the increase of pH. BSA has a molecular weight of ∼66 kDa, while the molecular weight of IgG is ∼150 kDa. These values indicate that a similar mole of individual proteins was adsorbed on the resin under their own optimized pH conditions, which may be related to the ligand density and distribution on resins. These results are consistent with the research by Tong et al.19 who studied similar adsorption processes in a column separation investigation. Research has shown that the optimized pH values for protein adsorption are usually around their individual isoelectric points because of strong hydrophobic interactions between proteins and HCIC ligands.20,21 However, with the addition of impurities with competitive adsorption phenomenon, purification conditions may be changed in order to achieve optimum performance, and this optimum condition may change when different impurities exist. To better understand the competitive adsorption behaviors for these two proteins under different conditions, it may be useful to study how pH, mass ratio, and salt can affect the competitive adsorption behaviors when the two proteins are loaded together. Meanwhile, the discrepancy of both adsorption capacity and optimum pH between IgG and

Q mC* Kd + C *

(1)

where Q* is the equilibrium adsorption capacity (mg/g resin), C* is the equilibrium protein concentration in the liquid (mg/ mL), Qm is the saturated adsorption capacity (mg/g resin), and Kd is the apparent dissociation constant (mg/mL). 2.3. Adsorption Kinetics. Adsorption kinetic studies were performed under similar conditions and parameters as those of static adsorption. A series of 40 mg of resin and 0.8 mL of protein solutions were mixed and shaken in a thermoshaker at 25 °C. After predefined time intervals the mixtures were filtered to obtain liquid phases. These liquid samples were analyzed by HPLC to acquire protein concentrations. The time course of protein concentration in the liquid phase was determined. 1354



Article

(BSA/IgG) mixture was only one-third of that of pure BSA (pH 5). With the increase of pH from 5 to 6, the adsorption of IgG increased dramatically, and it shows similar static adsorption from pH 6 to 8. Moreover, the increase of BSA in the protein mixture resulted in the decrease of IgG adsorption. However, at pH 8, the maximum adsorption capacity of BSA was lower than 20 mg/mL in protein mixtures, and the IgG adsorption seems not affected by the addition of BSA (Figure 2g,h). Figure 2 indicates that these proteins may compete for binding sites on resins, and the increase of one component can lead to the decrease of adsorption capacity of another. It is possible that based on the Vroman effect,22 BSA with smaller molecular size and higher mobility and concentration would reach and adsorb onto binding sites faster than IgG, which will later be replaced by larger proteins with higher affinity, that is, IgG.23 Therefore, adsorption kinetics was used to reveal the competitive binding process. 3.2. Adsorption Kinetics. Figure 3a shows the adsorption kinetics of BSA under conditions of BSA/IgG = 4:1 and pH 5− 8. These kinetic profiles clearly show the dynamic adsorption process of BSA on MEP HyperCel. The results show that

BSA may result in high capacity and adsorption selectivity of IgG to BSA. Figure 2 shows the adsorption isotherms of protein mixtures under different mass ratios and pH. The presence of another

Figure 2. Adsorption isotherms of binary BSA and IgG under different pH and mass ratios: (a) BSA, pH 5; (b) IgG, pH 5; (c) BSA, pH 6; (d) IgG, pH 6; (e) BSA, pH 7; (f) IgG, pH 7; (g) BSA, pH 8; (h) IgG, pH 8. Mass ratios: (■) single-component; (●) BSA/IgG = 4:1; (○) BSA/IgG = 3:1; (△) BSA/IgG = 2:1; (×) BSA/IgG = 1:1.

protein clearly affects the static adsorption processes of IgG/ BSA, and Figure 2 indicates that this competitive adsorption behavior is pH, protein concentration, and mass-ratio dependent. Figure 2 panels a and b show that BSA and IgG had similar adsorption capacity individually at pH 5. The adsorption capacity of both proteins declined with the addition of another component. For protein concentrations less than ∼2 mg/mL, the adsorption capacity of BSA under different IgG/BSA mass ratios was comparable to that of the pure BSA profile. Meanwhile, the adsorption capacity of IgG under same condition showed over 25% decrease as compared to that of pure IgG data. This may because the ligands were not fully occupied by these proteins when the protein concentration was less than ∼2 mg/mL, and pH 5 was a good adsorption condition for BSA. However, with the increase of IgG concentration, the BSA adsorption capacity decreased gradually. For example, the BSA adsorption capacity of the 1:1

Figure 3. Adsorption kinetics of BSA and IgG under different pH: (a) BSA, (b) IgG adsorption kinetic profiles; BSA/IgG = 4:1; initial protein concentration 10 mg/mL. (■) pH 5; (□) pH 6; (△) pH 7; (×) pH 8. (c) IgG fractions on resin: (q[IgG]/(q[IgG] + q[BSA])). Dotted data were measured and solid lines were fitting curves following the modified Linear Driving Force (LDF) model. 1355



Article

adsorption was the main process in the first few minutes for all pH conditions studied, and desorption gradually became the dominant process later on and last until the end of the experiments. Moreover, the maximum capacity appeared earlier with the increase of pH. For example, the curve of pH 8 started to decline after only 15 s, and there was no BSA detectable in the end of the experiment. The adsorption of protein in resin is a complex mass transport process and it can be affected by physical and chemical properties of resins, media, and proteins. A series of models have been proposed to study and predict protein adsorption kinetics in resins, such as pore diffusion and solid diffusion models, but for general curve fitting applications these models may show similar results.24 The Linear Driving Force (LDF) model is a simple empirical model (eq 2) ∂q(t ) = k(qmax − q(t )) ∂t

Table 1. Kinetic Fitting Parameters of BSA and IgG at Different pH BSA

(2)

(3)

where q(t) is the adsorption capacity at time t, qmax is the maximum adsorption capacity, and k is a constant. Good agreement between the LDF model and more rigorous diffusion models can be achieved when k = 15D/r2 (D is diffusivity and r is the radius of resins). This empirical model can greatly simplify the mathematical complexity and also is adequate for many practical applications.24 To use eq 3 to predict BSA adsorption behaviors, two adjustments are needed. (1) A time correction term tc is needed to compensate the time required for sample preparation before measurements; (2) the desorption process is also need to be included for BSA. The modified equation is as follows q(t ) = qads(1 − exp(kadst ′) − qdes(1 − exp(kdest ′)

qads

kads

qdes

kdes

qads

kads

5 6 7 8

85.51 66.30 28.23 31.22

0.85 0.31 0.13 0.07

54.62 57.68 26.85 30.12

0.12 0.06 0.13 0.13

22.70 30.96 28.11 25.84

0.11 0.11 0.17 0.27

be calculated as a function of time. Figure 3c indicates that pH is an important factor determining IgG fraction on resin, and it may take over 15 min for IgG to reach its maximum adsorption on the resin. A column separation study is a useful verification approach to further confirm the data profiles shown in this study.19 The separation study indicates that pH 7−8 combined with small flow rates or sufficient column length is a good separation condition to purify IgG from BSA/IgG mixtures. The adsorption kinetics was also studied under pH 5 where BSA and IgG had similar static adsorption capacities, with different mass−ratio protein mixtures. Similar to the results shown in Figure 3a, those in Figure 4a show that BSA can be quickly adsorbed under all mass ratio conditions, and within less than 5 min the maximum capacity was reached. The curves then show a decline of BSA adsorption capacity, which implies a competitive adsorption between BSA and IgG. Figure 4b shows that the IgG adsorption process was relatively slow compared to that of BSA, and the adsorption capacity is related to the mass percentage of IgG in the protein mixtures. The results of “IgG fraction on resin” in the figure give direct information about the final proportion of the two proteins bound on the resin. Figure 4c indicates that the maximum fraction of IgG adsorbed on the resin was less than 0.7 under pH 5. Therefore, Figure 3c and Figure 4c indicate that pH is an important factor that can dramatically affect the mass fraction of IgG adsorbed on the resin during competition adsorption. 3.3. Salt Addition. NaCl and (NH4)2SO4 are two widely used salts for protein separation and purification applications. These salts can precipitate proteins under certain concentration conditions. Meanwhile, the addition of these salts may affect the competitive adsorption process between BSA and IgG. Therefore, the precipitation of the two proteins was studied under experimental conditions in advance of adsorption studies, and no precipitation was found under the salt and protein concentrations studied. Figure 5 and Figure 6 show the adsorption isotherms of BSA and IgG with the addition of varied amounts of NaCl or (NH4)2SO4. The results show that the BSA adsorption capacity decreased dramatically when NaCl concentration increased (Figure 5a), while the adsorption capacity of IgG gradually increased (Figure 5b). The adsorption of BSA was almost not detectable with the addition of 0.75 or 1.0 M NaCl. Therefore, the IgG fraction on the resin can reach close to 1 when 1.0 M NaCl is added, which is significantly higher than that without NaCl under same conditions (∼0.4, see Figure 4c). However, Figure 6 shows slightly different results when (NH4)2SO4 was added. Although IgG showed the same trend with the increase of (NH4)2SO4 concentration, the BSA adsorption capacity showed a “U” shape change. The BSA adsorption capacity decreased after adding 0.25 M (NH4)2SO4; however, it increased with the further addition of (NH4)2SO4. IgG has a much larger molecular weight than BSA. Research has shown that electrostatic interactions play a dominant role in

which can be represented by the following equation by solving mass transfer differential equation: q(t ) = qmax (1 − exp(kt ))

IgG

pH

(4)

where qads is the adsorption capacity, qdes is the desorption capacity, kads is the adsorption parameter, and kdes is the desorption parameter. t′ = t − tc is the adjusted time. tc is a displacement factor which practically indicates the time required for the sample preparation and filtration. For experimental data obtained at longer times, this problem can be neglected. Meanwhile, a desorption term is applied because of the competitive adsorption between these two proteins. It was found that the amount of BSA adsorbed on the resin decreased after certain adsorption times. Therefore, the classical adsorption model is not suitable for fitting such data. Moreover, this phenomenon is likely indicating the existence of dynamic adsorption−desorption processes during the competitive adsorption. Figure 3b shows the adsorption kinetics of IgG, which has a gradual increase in the profiles, and the results show that pH did not have a significant effect on the adsorption of IgG. Equation 4 can also be used to fit the adsorption kinetics of IgG; however, as IgG did not show an obvious desorption process, the desorption part of eq 4 was neglected. The fitting curves in Figure 3a,b show that this modified LDF model can well fit the experimental data. The related fitting parameters are listed in Table 1. By comparing BSA and IgG adsorption kinetics shown in Figure 3a,b, the IgG fractions on resin (q[IgG]/(q[IgG] + q[BSA])), which indicates the changes of IgG percentage on the resin, can 1356



Article

Figure 5. Adsorption isotherms of BSA and IgG under different NaCl concentrations at pH 5. (a) BSA, (b) IgG; BSA/IgG = 4:1. (○) 0 M; (■) 0.25 M; (□) 0.5 M; (Δ) 0.75 M; (×) 1.0 M. Dotted data were measured and solid lines were fitting curves following the Langmuir model.

Figure 4. Adsorption kinetics of BSA and IgG under different BSA/ IgG mass ratios: (a) BSA, (b) IgG adsorption kinetic profiles; pH 5, initial protein concentration 10 mg/mL. (○) single-component; (■) BSA/IgG = 4:1; (□) BSA/IgG = 3:1; (△) BSA/IgG = 2:1; (×) BSA/ IgG = 1:1. (c) IgG fractions on resin: (q[IgG]/(q[IgG] + q[BSA])). Dotted data were measured and solid lines were fitting curves following the modified Linear Driving Force (LDF) model.

the BSA adsorption processes on HCIC resins. Meanwhile, hydrophobic interactions are the main adsorption mechanism for IgG binding onto MEP HyperCel.10,19,25 NaCl is a neutral salt which can behave as an electrostatic interaction screener but may not change the hydrophobic property of proteins within certain concentrations. Therefore, the addition of NaCl impedes the electrostatic interactions between BSA and the ligands, which results in the decrease of BSA adsorption capacity. However, the interaction between IgG and ligands was not much affected. The increase of adsorption capacity shown in Figure 5b was probably due to desorption of BSA that left more binding sites available for IgG, which is also a result of competitive adsorption.

Figure 6. Adsorption isotherms of BSA and IgG under different (NH4)2SO4 concentrations at pH 5. (a) BSA, (b) IgG; BSA/IgG = 4:1. (○) 0 M; (■) 0.25 M; (□) 0.5 M; (△) 0.75 M; (×) 1.0 M. Dotted data were measured and solid lines were fitting curves following the Langmuir model.

1357



Article

(NH4)2SO4 is a kosmotropic salt which can improve hydrophobic interactions between proteins and ligands, and it has been widely used in protein precipitation and hydrophobic interaction chromatography. Therefore, the addition of (NH4)2SO4 could facilitate the binding of IgG onto HCIC ligands. This is probably one reason for the increase of IgG adsorption as shown in Figure 6b. The adsorption of BSA on the ligands was likely due to the results of both electrostatic and hydrophobic interactions depending on (NH4)2SO4 concentration. When (NH4)2SO4 concentration was relatively low (∼0.25 M), electrostatic interactions still dominated, and (NH4)2SO4 has similar effect as NaCl, which leads to the decrease of BSA adsorption. With the increase of (NH4)2SO4 concentration, hydrophobic interactions gradually turn into action, which helps the adsorption of BSA on the resin. However, the maximum adsorption capacity of BSA after adding (NH4)2SO4 was lower than that without (NH4)2SO4. A detailed mechanism may need further studies using other techniques, such as Surface Plasmon Resonance. In summary, the addition of salts can improve the purification of IgG from BSA/IgG mixtures. Figure 7 shows that the adsorption kinetics of BSA and IgG with the addition of salts had similar profiles of that without salts (Figures 3 and 4). BSA showed maximum adsorption in less than 5 min, and then the desorption process overtook the adsorption process (Figure 7a). The addition of (NH4)2SO4 led to the decline of BSA adsorption, although 0.25 M (NH4)2SO4 showed lower adsorption capacity than that of 1.0 M. Figure 7c shows that the addition of salts resulted in an improvement of IgG fraction on resin under the experimental conditions, and the results showed that the addition of 0.25 M NaCl or (NH4)2SO4 had similar enhancing effects, which is better than 1.0 M (NH4)2SO4 or no salt conditions. 3.4. Discussion. Competitive adsorption in protein purification has been studied using different proteins mixtures in the literature,26,27 and techniques such as confocal laser scanning microscopy are used as a powerful imaging tool to qualitatively study competitive adsorption processes.28 Meanwhile, competitive adsorption or displacement of proteins on different material surfaces has been widely investigated since the 1960s, which is now commonly referred to as the Vroman effect.29 This effect has been discussed on solid surfaces with different charge properties30 and different protein pairs,23,31 and the mechanism has been discussed on a molecular level.32 However, this phenomenon is still not well understood, and there are typically three possible mechanisms to explain the competition process.29 Figure 8a shows a desorption−adsorption mode, in which BSA desorbs first and then IgG adsorbs onto the ligand. In the competitive binding process (Figure 8b), BSA is displaced by neighboring IgG attached on ligands. Figure 8c shows the third mode where a transient complex between BSA and IgG is first formed and then BSA gradually moves away from the ligand. In the end the ligands were occupied by IgG. It is difficult to confirm which mode actually happened in this study. The competitive exchange mode can be easily achieved on material surfaces, but it may require high ligand density of resins in chromatography. The transient complex formation process may need a longer period for IgG to be absorbed onto the ligands. However, the kinetic profiles of BSA adsorption show that it can usually happened within 5 min. Therefore, the desorption− adsorption mode may be the most possible mechanism of the BSA/IgG mixture adsorption processes.

Figure 7. Adsorption kinetics of BSA and IgG under different salt concentrations at pH 5. (a) BSA, (b) IgG; BSA/IgG = 4:1; (○) 0 M; (■) 0.25 M (NH4)2SO4; (×) 1 M (NH4)2SO4; (▽) 0.25 M NaCl. (c) IgG fractions on resin: (q[IgG]/(q[IgG] + q[BSA])). Dotted data were measured and solid lines were fitting curves following the modified Linear Driving Force (LDF) model.

4. CONCLUSIONS A commercial HCIC resin MEP HyperCel was used to investigate competitive adsorption processes of BSA and IgG in order to better understand the adsorption behaviors and optimize IgG separation efficiency from protein mixtures with HCIC resins. The adsorption isotherms and dynamic binding processes were determined under different media pH and mass ratio of BSA/IgG mixtures. The results showed that pH was an important factor on determining the protein adsorption capacity of the resin, and the increase of one component in the mixture would result in the decrease of adsorption capacity of another component, as an indication of competitive adsorption. Kinetic results showed that BSA can be adsorbed on the ligands quicker than IgG but part of the adsorbed BSA 1358



Article

Figure 8. A schematic diagram illustrating three competitive adsorption processes: (a) desorption/adsorption processes; (b) competitive binding processes; (c) displacement through the formation of transient complexes. Adapted with permission from ref 29. Copyright 2012 Elsevier. (6) Li, R.; Dowd, V.; Stewart, D. J.; Burton, S. J.; Lowe, C. R. Design, synthesis, and application of a protein a mimetic. Nat. Biotechnol. 1998, 16, 190−195. (7) Bak, H.; Thomas, O. R. T. Evaluation of commercial chromatographic adsorbents for the direct capture of polyclonal rabbit antibodies from clarified antiserum. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2007, 848, 116−130. (8) Tong, H.-F.; Lin, D.-Q.; Gao, D.; Yuan, X.-M.; Yao, S.-J. Caprylate as the albumin-selective modifier to improve IgG purification with hydrophobic charge-induction chromatography. J. Chromatogr. A 2013, 1285, 88−96. (9) Ghose, S.; Hubbard, B.; Cramer, S. M. Protein interactions in hydrophobic charge induction chromatography (HCIC). Biotechnol. Prog. 2005, 21, 498−508. (10) Arakawa, T.; Kita, Y.; Sato, H.; Ejima, D. MEP chromatography of antibody and Fc-fusion protein using aqueous arginine solution. Protein Expression Purif. 2009, 63, 158−163. (11) Guerrier, L.; Girot, P.; Schwartz, W.; Boschetti, E. New method for the selective capture of antibodies under physiolgical conditions. Bioseparation 2000, 9, 211−221. (12) Guerrier, L.; Flayeux, I.; Boschetti, E. A dual-mode approach to the selective separation of antibodies and their fragments. J. Chromatogr., Biomed. Appl. 2001, 755, 37−46. (13) Morrison, C. J.; Gagnon, P.; Cramer, S. M. Purification of monomeric mAb from associated aggregates using selective desorption chromatography in hydroxyapatite systems. Biotechnol. Bioeng. 2011, 108, 813−821. (14) Carta, G.; Lewus, R. Film Model Approximation for Multicomponent Adsorption. Adsorption 2000, 6, 5−13. (15) Martin, C.; Iberer, G.; Ubiera, A.; Carta, G. Two-component protein adsorption kinetics in porous ion exchange media. J. Chromatogr. A 2005, 1079, 105−115. (16) Shi, Q.-H.; Shi, Z.-C.; Sun, Y. Dynamic behavior of binary component ion-exchange displacement chromatography of proteins visualized by confocal laser scanning microscopy. J. Chromatogr. A 2012, 1257, 48−57. (17) El-Sayed, M. M. H.; Chase, H. A. Confocal microscopy study of uptake kinetics of α-lactalbumin and β-lactoglobulin onto the cationexchanger SP Sepharose FF. J. Sep. Sci. 2009, 32, 3246−3256. (18) Teske, C. A.; Von Lieres, E.; Schröder, M.; Ladiwala, A.; Cramer, S. M.; Hubbuch, J. J. Competitive adsorption of labeled and native protein in confocal laser scanning microscopy. Biotechnol. Bioeng. 2006, 95, 58−66. (19) Tong, H.-F.; Lin, D.-Q.; Yuan, X.-M.; Yao, S.-J. Enhancing IgG purification from serum albumin containing feedstock with hydro-

would be gradually displaced by IgG as a result of competitive adsorption. A modified Linear Driving Force model was used, which showed good fitting results of the adsorption kinetic profiles. The effect of salts on the adsorption process was also studied, and the results showed that both NaCl and (NH4)2SO4 can enhance the adsorption selectivity of IgG on MEP HyperCel, although the effects and mechanism of these two salts were different based on the electrostatic and hydrophobic interactions. In addition, the desorption−adsorption mode may be suitable to explain the competitive adsorption process of IgG and BSA.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

This work was supported by the National Natural Science Foundation of China and the Zhejiang Provincial Natural Science Foundation of China. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS Mr. Ferdinand Schimpf was a master student from Technische Universität München. REFERENCES

(1) Menachem, A.; Chapman, J.; Deri, Y.; Pick, C.; Katzav, A. Immunoglobulin-Mediated Neuro-Cognitive Impairment: New Data and a Comprehensive Review. Clin. Rev. Allergy Immunol. 2013, 45, 248−255. (2) Reichert, J. M. Antibodies to watch in 2014: Mid-year update. mAbs 2014, 6, 799−802. (3) Li, F.; Vijayasankaran, N.; Shen, A.; Kiss, R.; Amanullah, A. Cell culture processes for monoclonal antibody production. mAbs 2010, 2, 466−479. (4) Low, D.; O’Leary, R.; Pujar, N. S. Future of antibody purification. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2007, 848, 48−63. (5) Hober, S.; Nord, K.; Linhult, M. Protein A chromatography for antibody purification. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2007, 848, 40−47. 1359



Article

phobic charge-induction chromatography. J. Chromatogr. A 2012, 1244, 116−122. (20) Zhao, G.; Peng, G.; Li, F.; Shi, Q.; Sun, Y. 5-Aminoindole, a new ligand for hydrophobic charge induction chromatography. J. Chromatogr. A 2008, 1211, 90−98. (21) Lin, D.-Q.; Tong, H.-F.; Wang, H.-Y.; Yao, S.-J. Molecular Insight into the Ligand−IgG Interactions for 4-Mercaptoethyl-pyridine Based Hydrophobic Charge-Induction Chromatography. J. Phys. Chem. B 2012, 116, 1393−1400. (22) Vroman, L.; Adams, A.; Fischer, G.; Munoz, P. Interaction of high molecular weight kininogen, factor XII, and fibrinogen in plasma at interfaces. Blood 1980, 55, 156−159. (23) Holmberg, M.; Stibius, K.; Larsen, N.; Hou, X. Competitive protein adsorption to polymer surfaces from human serum. J. Mater. Sci.: Mater. Med. 2008, 19, 2179−2185. (24) Carta, G.; Jungbauer, A., Adsorption Kinetics. In Protein Chromatography; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2010; pp 161−199. (25) Arakawa, T.; Futatsumori-Sugai, M.; Tsumoto, K.; Kita, Y.; Sato, H.; Ejima, D. MEP HyperCel chromatography II: Binding, washing and elution. Protein Expression Purif. 2010, 71, 168−173. (26) Xu, X.; Lenhoff, A. M. Binary adsorption of globular proteins on ion-exchange media. J. Chromatogr. A 2009, 1216, 6177−6195. (27) Traylor, S. J.; Xu, X.; Lenhoff, A. M. Shrinking-core modeling of binary chromatographic breakthrough. J. Chromatogr. A 2011, 1218, 2222−2231. (28) Ljunglöf, A.; Hjorth, R. Confocal microscopy as a tool for studying protein adsorption to chromatographic matrices. J. Chromatogr. A 1996, 743, 75−83. (29) Hirsh, S. L.; McKenzie, D. R.; Nosworthy, N. J.; Denman, J. A.; Sezerman, O. U.; Bilek, M. M. M. The Vroman effect: Competitive protein exchange with dynamic multilayer protein aggregates. Colloids Surf., B 2013, 103, 395−404. (30) Lassen, B.; Malmsten, M. Competitive Protein Adsorption at Plasma Polymer Surfaces. J. Colloid Interface Sci. 1997, 186, 9−16. (31) Huetz, P.; Ball, V.; Voegel, J. C.; Schaaf, P. Exchange Kinetics for a Heterogeneous Protein System on a Solid Surface. Langmuir 1995, 11, 3145−3152. (32) Jung, S.-Y.; Lim, S.-M.; Albertorio, F.; Kim, G.; Gurau, M. C.; Yang, R. D.; Holden, M. A.; Cremer, P. S. The Vroman Effect: A Molecular Level Description of Fibrinogen Displacement. J. Am. Chem. Soc. 2003, 125, 12782−12786.

1360


Binary Adsorption Processes of Albumin and Immunoglobulin on

Recommend Documents