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5-Aminobenzimidazole as new hydrophobic charge-induction ligand for expanded bed adsorption of bovine IgG
Journal of Chromatography A, 1425 (2015) 97–105
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
5-Aminobenzimidazole as new hydrophobic charge-induction ligand for expanded bed adsorption of bovine IgG Wei Shi a , Dong-Qiang Lin a,∗ , Hong-Fei Tong a , Jun-Xian Yun b , Shan-Jing Yao a a Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China b State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China
a r t i c l e
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Article history: Received 6 August 2015 Received in revised form 24 September 2015 Accepted 16 October 2015 Available online 24 October 2015 Keywords: Expanded bed adsorption Hydrophobic charge-induction chromatography 5-Aminobenzimidazole Bovine whey IgG
a b s t r a c t Expanded bed adsorption (EBA) can capture target proteins directly from unclarified feedstock without prior solid–liquid separation. Hydrophobic charge-induction chromatography (HCIC) is a promising technology for biomolecule separation with high capacity, good selectivity and relatively low cost without the pretreatment of dilution or salt addition. In this work, EBA and HCIC were combined to develop a new separation technology, hydrophobic charge-induction EBA. Two HCIC ligands, 4-mercapto-ethyl-pyridine (MEP) and 5-aminobenzimidazole (ABI), were coupled onto agarose beads containing tungsten carbide to prepare the resins for EBA, named T-MEP and T-ABI, respectively. The static adsorption and dynamic binding behaviors of bovine IgG (bIgG) were investigated. Two resins had similar saturated adsorption capacities and salt-tolerant properties, but T-ABI showed higher dynamic binding capacity than T-MEP, indicating that ABI ligand was more suitable for EBA. The performances in expanded bed were verified. With the protein mixture (2 mg/ml bIgG and 10 mg/ml bovine serum albumin) as the model feedstock, the effects of loading and elution pH, expansion factor and loading volume on the separation performance of bIgG were evaluated. Finally, T-ABI EBA was used to separate bIgG directly from bovine whey with optimized operation conditions. The purity and recovery of bIgG reached 90.6% and 78.2%, respectively. The purification factor was about 19.3. The results demonstrated that the combination of HCIC and EBA would be a potential platform for antibody capture with less feedstock pretreatments, high efficiency and relatively low cost. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Expanded bed adsorption (EBA) was developed in 1990s as an innovative technology that allows capturing target proteins directly from unclarified feedstock, e.g., culture suspensions and cell homogenates [1–3], which combines solid–liquid separation with an adsorption step in a single-unit operation [4,5]. When the mobile phase is pumped upward through the column, the resin beads with an appropriate distribution of size/density can form a perfectly classified fluidized bed with low back-mixing. Cell or cell debris can pass through the voidage between resin beads without blocking the column and the target can be adsorbed by the resin. Obviously, EBA technology can reduce operational time and increased overall yield with less requirements for capital investment and consumables. In general, specially designed resins are
∗ Corresponding author. E-mail address: [email protected] (D.-Q. Lin). http://dx.doi.org/10.1016/j.chroma.2015.10.055 0021-9673/© 2015 Elsevier B.V. All rights reserved.
critical for EBA operation, which have a significant influence on the feasibility and the overall performance of the process. During the past 20 years, series of matrices have been developed for EBA. For example, Streamline and Streamline Direct are two series of the well-known commercial resins for EBA developed by GE Healthcare (Uppsala, Sweden), which are based on cross-linked agarose containing a crystalline quartz core or stainless steel power as the densifier. These resins show some applications for protein separation, but new series of resins are still needed to improve the performance of EBA. The functional ligands coupled on the resin beads are an important factor to determine the adsorption efficiency of EBA process. The most common applications for EBA are ion-exchange and hydrophobic interaction resins. However, due to the complexity of biomolecular structure, the separation selectivity of these two kinds of binding modes is normally limited. In addition, the conductivity of the feedstock should be considered and some pretreatments have to be done, such as the dilution for ion exchange and the addition of salt for hydrophobic interaction separations,
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Fig. 1. Preparation scheme of two HCIC resins T-MEP and T-ABI.
which certainly increase the cost and process time. Hydrophobic charge-induction chromatography (HCIC) with dual-mode ionizable ligands is new protein separation method [6], which often shows high capacity at a wide range of salt concentration. HCIC ligands, such as 4-mercapto-ethyl-pyridine (MEP) with the pKa of 4.8, show a typical pH-dependent property, from non-charge to positive charges with the decrease of pH. The target protein can be adsorbed on the uncharged ligand at neutral pH by the hydrophobic interactions, and desorbed by the electrostatic repulsion between target protein and the charged ligand at acidic condition. HCIC has been applied to the purification of antibodies and other proteins, which showed a potential alternative to Protein A chromatography [7–10]. Xia et al. [11] coupled three HCIC ligands, MEP, 2-mercapto1-methyl-imidazole (MMI) and 2-mercapto-benzimidazole (MBI) onto the macroporous cellulose-tungsten carbide composite beads, and used for EBA separation of immunoglobulin Y (IgY) from egg yolk. The results demonstrated that HCIC in expanded bed mode is a potential technology for antibody separation both in high efficiency and high productivity. However, due to weak hydrophobic interactions between target protein and the ligands used, the dynamic binding capacities were relatively low, especially for high operation velocity for EBA. Therefore, new HCIC ligands should be developed to enhance the binding forces and improve the applications for EBA processes. In the present work, the tungsten-carbide-densified agarose beads were used as the matrix for EBA. The agarose beads containing tungsten carbide as the densifier have relatively high density (2.8–3.2 g/ml) compared to the beads with quartz glass (Streamline series) and steel core (Streamline Direct series), which can be used for high superficial velocity and lead to high production. Two HCIC ligands, MEP and 5-aminobenzimidazole (ABI), were coupled onto the matrix to prepared new resins for EBA. The former represents the typical HCIC ligand, and the latter is newly-designed ligand based on the molecular simulation [12]. The structures of MEP and ABI are shown in Fig. 1. Compared with MEP with a pyridine ring, ABI had two cyclic structures including one benzene ring and one imidazole ring, which might improve the hydrophobic binding with target protein. The pKa of ABI is 6.5, resulting in milder pH condition for the charge-induction effect and protein elution. In addition, the results of molecular simulation showed that ABI could bind onto the consensus binding site of Fc domain of IgG molecule in a favorable way [12], so ABI was considered as a new HCIC
ligand for antibody purification. Therefore, two ligands, MEP and ABI, would be evaluated and compared for EBA process in this work. Firstly, the adsorption behaviors of bovine IgG (bIgG) on two resins would be investigated with batch and frontal adsorption experiments. The effects of pH and salt on the static and dynamic binding capacity would be focused. Then, the separation of bIgG from the bovine serum albumin (BSA)-containing feedstock would be investigated, and the bed expansion and liquid mixing in expanded bed were evaluated. Finally, EBA process was developed to separate bIgG from bovine whey. The potential of the application of new HCIC resins for antibody purification with EBA process would be discussed. 2. Materials and methods 2.1. Materials ABI and MEP were purchased from J&K technology Co., Ltd. (China). Crosslinked agarose beads containing tungsten carbide were prepared with similar methods as published in our previous work [11,13]. Bovine serum ␥-globulin (bovine IgG, electrophoresis purity 99.0%) was purchased from Merck KGaA (Darmstadt, Germany). BSA (67 kDa) was purchased from Sigma (Milwaukee, WI, USA). Bovine milk was obtained from local resource. All other chemical materials were of analytical grade and were purchased commercially. 2.2. Preparation of resins The preparation procedure was similar to that published by Gao et al. [13] and Xia et al. [11]. The matrices were activated with allyl bromide at the basic condition and brominated by Nbromosuccinimide (NBS), then coupled with HCIC ligands, MEP or ABI, under proper conditions. Typically, 10 g drained agarose beads were mixed with 4 ml allyl bromide and 5 g NaOH in 11 ml 20% dimethyl sufoxide (DMSO), and the mixture was continuously agitated at 180 rpm and 50 ◦ C for 24 h. The allyl-activated matrices were washed with deionized water and mixed with 10 ml H2 O, 10 ml acetone, and 3 molar excess of NBS over the allyl groups and agitated at 180 rpm and 30 ◦ C for 1 h. Then, the brominated matrices were mixed with 3 molar excess of MEP or ABI over the allyl groups in 10 ml 1 M Na2 CO3 (pH ∼11.7), and agitated at 180 rpm
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and 30 ◦ C for 12 h. Finally the beads were washed with 0.1 M HCl, 0.1 M NaOH and deionized water, and the resins were stored in 20% (v/v) ethanol. Two kinds of HCIC resins were prepared, namely T-MEP and T-ABI, respectively. 2.3. Adsorption equilibrium experiments The static adsorption behaviors of bIgG on two resins were measured by batch adsorption equilibrium experiments with the procedure as the references [14,15]. 20 mM sodium acetate buffer (pH 5.0), 20 mM phosphate buffer (pH 6.0, pH 7.0 and pH 8.0) and 20 mM glycine buffer (pH 9.0 and pH 10.0) were used as the liquid phases for different pHs. After the adsorption reached equilibrium, the protein concentration in the supernatant was determined at 280 nm with One Drop Spectrophotometer (Nanjing WuYi Technology Co., Ltd., Nanjing, China). The adsorption isotherm was fitted with the Langmuir equation, and the saturated adsorption capacity (Qm ) and the dissociation constant (Kd ) were obtained. Ammonium sulfate and sodium chloride were also added into the buffer (0, 0.2, 0.4, 0.6 and 0.8 M), respectively, and used as the liquid phase to investigate the influence of salt concentration on the adsorption of bIgG on two resins. 2.4. Evaluation with packed bed 5-mm I.D. column (Tricorn 5/100, GE Healthcare, Uppsala, Sweden) was packed with 2.0 ml resins (about 10 cm settled bed height). The dynamic binding capacity was investigated through frontal adsorption experiments. The column was equilibrated with the equilibrium buffer. Then, bIgG solution (2 mg/ml) was loaded into the column at the superficial linear velocity of 200, 400 or 600 cm/h. The protein concentration in the effluent was monitored by absorbance at 280 nm with a UV monitor LC3000 (Beijing Chuangxintongheng Science & Technology Co., Ltd., Beijing, China). The dynamic binding capacity at 10% breakthrough (Q10% ) was calculated as described by Griffith et al. [16]. The separation of bIgG from BSA containing feedstock was evaluated with the same column and the ÄKTA explorer 100 system (GE Healthcare, Uppsala, Sweden). Loading and elution pH were screened. 20 mM phosphate buffer (pH 7.0 and pH 8.0) and 20 mM glycine buffer (pH 9.0) were used as the loading buffer, and 20 mM acetate buffer (pH 3.0, 3.5, 4.0 and 4.5) were used as the elution buffer. The protein mixture containing 2 mg/ml bIgG and 10 mg/ml BSA was used as the loading feedstock. The flow rate was 0.5 ml/min. The column was equilibrated with the loading buffer and then 2 ml protein mixture (corresponding to 1-fold column volume) was loaded. After sample loading, the column was washed by the loading buffer and then bIgG was eluted with the acetate buffer. Finally, the column was regenerated with 0.1 M NaOH and re-equilibrated with the loading buffer for the next test. The chromatographic run was monitored on-line at 280 nm. The fractions were collected and analyzed with the SEC-HPLC and SDS-PAGE. 2.5. Characteristics in expanded bed 1 cm-diameter home-made column was used, and 13.5 ml resins (about 19 cm settled bed height, H0 ) were packed into the column. All experiments were performed at room temperature and the proper column vertical alignment was assured. The bed was expanded with 20 mM sodium phosphate buffer (pH 8.0) and the equilibrium state was reached after about 20–30 min of expansion and a stable bed height was obtained. The expanded bed height (H) was measured three times for one flow rate, and then the mean value was used to calculate the expansion factor (H/H0 ). Six expansion factors (1.6, 1.8, 2.0, 2.2, 2.4 and 2.6) were tested.
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Liquid mixing in expanded bed was evaluated by the measurement and analysis of residence time distribution (RTD). 0.5 ml acetone solution (10%, w/w) was used as the tracer. The response signal at the outlet was detected by the UV detector (LC3000, Beijing Chuangxintongheng Science & Technology Co., Ltd., Beijing, China). The RTD test for each sampling port was repeated 3–4 times. The axial dispersion coefficient (Dax ) was calculated based on the RTD curves [17,18]. 2.6. Frontal adsorption experiments in expanded bed The dynamic binding capacity of bIgG in expanded bed was determined by frontal adsorption experiments. 1 cm-diameter column and 13.5 ml resins (about 19 cm settled bed height) were used. The bed was firstly expanded with 20 mM sodium phosphate buffer (pH 8.0) and the equilibrium state was reached after 20–30 min. Four expansion factors (1.8, 2.0, 2.2 and 2.4) were tested. 2 mg/ml bIgG solution was then pumped into the column. The protein breakthrough at outlet was detected at 280 nm with LC3000. The dynamic binding capacity at 10% breakthrough was calculated as mentioned above. 2.7. Separation of bIgG from protein mixture with expanded bed 1 cm-diameter column and 13.5 ml resins (about 19 cm settled bed height) were used. The protein mixture containing 2 mg/ml bIgG and 10 mg/ml BSA was used as the loading feedstock as mentioned above. The influences of expansion factor (1.8, 2.0, 2.2 and 2.4) and loading volume (1, 2, 3 and 4 folds of settled bed volume) were investigated with the expanded bed system as mentioned above. The bed was expanded and equilibrated with 20 mM sodium phosphate buffer (pH 8.0) until a stable bed height was obtained. The protein mixture was loaded into the column. Then, the column was washed with the loading buffer to remove unbound proteins. After that, bIgG was eluted with 20 mM acetate buffer (pH 3.5). Finally, the column was regenerated with 0.1 M NaOH and reequilibrated with the loading buffer. The fractions were collected and analyzed with the SEC-HPLC and SDS-PAGE. The EBA process was repeated 3 times, and the average recovery and purity of bIgG were obtained. 2.8. Separation of bIgG from bovine whey with expanded bed Bovine whey was prepared as published previously [19]. The main processes were as follows. Bovine milk was centrifuged for 30 min at 5000 rpm to remove fat, and then adjusted to pH 4.7 by the addition of 5 M HCl. Afterwards, casein was precipitated from bovine milk by stirring at 35 ◦ C for 50 min, and bovine whey was obtained by centrifugation. The pH was adjusted to 8.0 with 2 M NaOH for the further EBA tests. The concentration and purity of bIgG in whey were analyzed with SEC-HPLC. Same expanded bed as mentioned above was used for bIgG separation. For bIgG separation from bovine whey, the bed with 13.5 ml T-ABI resins was expanded to 2.0 expansion factor with 20 mM sodium phosphate buffer (pH 8.0). 40 ml bovine whey was loaded. After sample loading, the column was washed with the equilibrium buffer. Then bIgG was eluted with 20 mM acetate buffer (pH 3.5). The column was regenerated with 0.1 M NaOH and re-equilibrated with the equilibrium buffer. The fractions were collected during the process and analyzed with the SEC-HPLC and SDS-PAGE to determine the recovery and purity of bIgG. 2.9. SEC-HPLC analysis The analytical SEC-HPLC was performed on LC3000 system (Beijing Chuangxintongheng Science & Technology Co., Ltd.,
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Beijing, China) with the TSK G3000SWXL column (7.8 mm × 30.0 cm, TOSOH, Japan). 0.1 M sodium phosphate buffer (pH 6.7, containing 0.1 M Na2 SO4 ) was used as the mobile phase. The buffer was filtrated with 0.22 m membrane and degassed before use. The flow rate was 0.5 ml/min. The purity of bIgG was defined as the percentage of the peak area of bIgG monomer to the total integrated peak areas. The recovery of bIgG was calculated as the percentage of bIgG monomer in the elution fraction to that in the loading sample during the separation process. 2.10. SDS-PAGE analysis The sample and fractions collected during the separation process were analyzed by 12% non-reducing SDS-PAGE. Protein migration was performed under 180 V for 50 min. The gel was stained with Coomassie Blue R-250 and destained. The Gel Doc 2000 imaging system (Bio-Rad, Hercules, CA, USA) was used to image the protein gel. 3. Results and discussion 3.1. Preparation of T-MEP and T-ABI resins Two HCIC resins, T-MEP and T-ABI, with tungsten carbideagarose composite beads as the matrix and MEP/ABI as the functional ligand were prepared with the route as shown in Fig. 1. The agarose beads were activated with allyl bromide firstly, and the density of allyl group on the activated bead was about 125 mol/ml resin. After brominated by NBS, the ligand coupling reaction was conducted under alkali condition as published previously [11,13]. The ligand densities of MEP and ABI on the resins prepared were 86 and 76 mol/ml resin, respectively. The results indicated that the preparation procedure was suitable, and the coupling efficiencies of two ligands were similar. 3.2. Adsorption equilibrium of bIgG on T-MEP and T-ABI resins The pH-dependent and salt-tolerant adsorption behaviors are typical characteristics of HCIC resins. Thus, it is important to investigate the influences of pH and salt concentration on the adsorption capacity of two new resins firstly. The batch experiments were adopted to study the static adsorption behaviors of bIgG. The adsorption isotherms of bIgG with T-MEP and T-ABI at different pH values and salt concentrations were determined. As shown in Fig. 2 and Table 1, pH had a significant influence on the adsorption of bIgG on both resins. For T-MEP, the highest adsorption capacities were found at pH 9.0 with the Qm of 86.45 mg/ml resin, and Kd was 0.30 mg/ml. The Qm values at pH 7.0 and 8.0 were comparable, i.e. 75.71 and 81.68 mg/ml resin, respectively. When the pH decreased to 5.0, the adsorption capacity of bIgG decreased remarkably. For T-ABI, the results were similar to Table 1 Equilibrium parameters Qm and Kd of bIgG adsorption with T-MEP and T-ABI at various pHs. pH
5.0 6.0 7.0 8.0 9.0 10.0
T-MEP
T-ABI
Qm (mg/ml resin)
Kd (mg/ml)
Qm (mg/ml resin)
Kd (mg/ml)
40.72 70.72 75.71 81.68 86.45 76.90
0.87 0.57 0.49 0.39 0.30 0.38
37.78 65.23 66.55 74.68 73.54 60.79
0.52 0.31 0.25 0.23 0.25 0.36
Fig. 2. The adsorption isotherms of bIgG with T-MEP (a) and T-ABI (b) at different pH values. (䊉) pH 5.0; () pH 6.0; () pH 7.0; () pH 8.0; () pH 9.0; () pH 10.0.
that of T-MEP. High adsorption capacities and low Kd values were also found at pH 7.0, 8.0 and 9.0. In addition, the adsorption capacity at pH 5.0 was quite low as well. Compared with T-MEP, T-ABI showed slightly lower adsorption capacities and Kd values. This might be due to relatively lower ligand density of T-ABI but stronger hydrophobic interactions of ABI ligand. For both resins, pH 7.0–9.0 would be suitable for bIgG adsorption and the acidic pH could be used for the elution. The effects of (NH4 )2 SO4 and NaCl addition on the adsorption equilibrium of bIgG with T-MEP and T-ABI at pH 8.0 are shown in Figs. S1 and S2 (supporting information), and the equilibrium parameters Qm and Kd are listed in Tables S1and S2 (supporting information). It was found that some adsorption isotherms did not reach the plateaus, especially for high salt concentration, so the fitted Qm values would be not suitable to describe adsorption ability. As suggested by Sun and coworkers [20–23], the adsorbed protein density (Qc ) at an equilibrium liquid-phase concentration calculated by Langmuir equation was introduced. The Qc values at the equilibrium liquid-phase concentration of 5 mg/ml were calculated and compared in Fig. 3. It could be found that two resins had similar “U” shape trends with the increase of salt concentration. Qc of bIgG declined slightly when salt concentration increased from 0 to 0.2 M. Then the adsorption capacity increased gradually with the increase of salt concentration from 0.2 to 0.8 M. For high salt concentration, Qc values were even higher than that without salt addition.
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Fig. 4. Comparison of Q10% with T-MEP and T-ABI in packed bed at different flow velocities.
Fig. 3. The Qc s of bIgG with T-MEP (a) and T-ABI (b) at pH 8.0 and the equilibrium liquid-phase concentration of 5 mg/ml as a function of different ammonium sulfate and sodium chloride concentrations.
The decline of the adsorption capacity for both resins at 0.2 M (NH4 )2 SO4 or NaCl might be due to the impact on the hydrogen bonds between HCIC ligand and bIgG under low salt concentration. When salt concentration increased continuously, salting-out effect would lead to the exposure of hydrophobic area on the surface of protein and improve the hydrophobic interactions between HCIC ligand and bIgG, resulting in the increase of adsorption capacity. In general, the adsorption capacities of T-MEP and T-ABI did not change significantly with the change of salt concentration, which indicated both resins had good property of salt tolerance.
ligand could provide stronger hydrophobic interactions with target IgG molecule, which certainly fasten the mass transfer in the pores and improve the rate of protein adsorption. Therefore, compared with T-MEP, T-ABI was more suitable for application for EBA with high flow velocity, and would be further studied for the applications of protein separation and EBA tests. T-ABI resin was challenged to purify bIgG from the protein mixture of bIgG and BSA with packed bed. The feedstock contained 2 mg/ml bIgG and 10 mg/ml BSA. The loading and elution pH were screened to optimize the separation efficiency. The chromatographic results at different loading pH conditions are shown in Fig. 5. With the sample loading at pH 7.0 and elution at pH 4.0, the recovery and purity of bIgG were 70.2% and 73.4%, respectively. When the loading pH increased to pH 8.0, the purity of bIgG decreased slightly (72.4%), but the recovery increased obviously (82.2%). At pH 9.0, bIgG had the highest purity of 76.8%, while the recovery decreased to 74.4%. Considering the recovery and purity together, pH 8.0 was chosen as the suitable loading pH for following separation experiments. The elution pH was optimized further, and the results are shown in Fig. 6. It was found that the elution peak heights increased with the decrease of elution pH from 4.5 to 3.0, indicating that the elution of bIgG was enhanced apparently at acidic condition. The recoveries of bIgG were 61.9%, 82.2%, 92.3% and 94.3% for the elution pH of 4.5, 4.0, 3.5 and 3.0, respectively. Highest purity of bIgG (90.3%) was found at the elution pH 4.5, while a relatively low purity of bIgG (67.6–72.4%) was found for the elution of pH 3.0–4.0. Considering the purity and recovery together, the elution at pH 3.5 would be suitable for bIgG separation from the BSA containing feedstock. 3.4. Expansion characteristics and liquid mixing in expanded bed
3.3. Dynamic binding capacity and protein separation in packed bed Dynamic binding capacities of bIgG with two resins at different flow velocities were evaluated by the frontal adsorption experiments in packed bed. The dynamic binding capacities at 10% breakthrough, Q10% , are compared in Fig. 4. Although the static adsorption capacities of T-ABI and T-MEP were similar, TABI had higher Q10% than T-MEP. With the increase of flow velocity from 200 to 600 cm/h, Q10% of T-ABI decreased from 15.15 to 6.74 and 5.21 mg/ml resin. For T-MEP, Q10% was only 5.65 mg/ml resin at 200 cm/h and decreased to 3.60 mg/ml resin at 400 cm/h and 2.53 mg/ml resin at 600 cm/h. The results indicated further that ABI
Expansion characteristic is one of the most important factors for EBA process, which affects greatly the adsorption performance. Normally the expansion factor is controlled at the range of 1.5–3.0 [24–28]. When the expansion factor is too low, the bed could not be expanded adequately. The excessive fluidization and the loss of small resin beads would be found when the expansion factor is too high. Therefore, based on the tungsten carbide-agarose composite beads used in the present work, the expansion factors of 1.6–2.6 were tested. The results are shown in Fig. 7. Due to high wet density of T-ABI resin with tungsten carbide as the densifier, high operation velocity (550–1355 cm/h) was needed for the expansion factors of 1.6–2.6. For the normal expansion factor of 2.0 for EBA process,
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Fig. 5. Chromatograms of bIgG separation at different loading pHs with T-ABI packed bed. Breakthrough (BT) with 20 mM phosphate buffer (pH 7.0, pH 8.0) and 20 mM glycine buffer (pH 9.0); elution with 20 mM acetate buffer (pH 4.0); CIP with 0.1 M NaOH. The flow rate was 0.5 ml/min.
the operation velocity was 889 cm/h with T-ABI, which was much higher than some commercial EBA resins, such as 264 cm/h for Streamline series and 517 cm/h for Streamline Direct series [29,30]. Liquid mixing in the expanded bed is an important property for evaluating the bed stability and protein adsorption. The liquid mixing and bed stability were evaluated with RTD tests at different expansion factors. The theoretical plate number and Dax were calculated. Small Dax means lower back mixing and a more stable bed. Dax as the function of flow velocity is shown in Fig. 7. It was found that the Dax increased with the increase of flow velocity, and all Dax values kept at a level of 10−5 m2 /s. Even for high flow velocity of about 1300 cm/h at the expansion factor of 2.6, the bed was still quite stable to meet the requirement of expanded bed operation.
3.5. Dynamic binding capacity in expanded bed The dynamic binding behaviors of bIgG with T-ABI in expanded bed at varying flow velocities were evaluated by the front adsorption experiments. As shown in Fig. 8, the breakthrough curves at flow velocities of 711, 889, 1050 and 1203 cm/h (corresponding to expansion factors 1.8, 2.0, 2.2 and 2.4, respectively) were quite similar. It was found that the Q10% s at varying flow velocities were close to each other (4.53 mg/ml resin for EF 1.8, 5.41 mg/ml resin for EF 2.0, 5.49 mg/ml resin for EF 2.2 and 5.10 mg/ml resin for EF 2.4), indicating that the expanded bed was quite stable. Q10% values in expanded bed were also near that in packed bed at 600 cm/h, which indicated that ABI ligand had a strong ability to capture bIgG
Fig. 6. Chromatograms of bIgG separation at different elution pHs with T-ABI packed bed. Breakthrough (BT) with 20 mM phosphate buffer (pH 8.0); elution with 20 mM acetate buffer (pH 3.0, 3.5, 4.0 and 4.5); CIP with 0.1 M NaOH. The flow rate was 0.5 ml/min.
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Fig. 7. Expansion factor and Dax as the function of flow velocity in expanded bed.
and a good protein adsorption could be maintained even for high flow velocity as 1000 cm/h. Compared different flow velocities, the operations at 889 cm/h (expansion factor of 2.0) and 1050 cm/h (expansion factor of 2.2) reached the highest dynamic binding capacities, which would be most suitable for EBA applications. 3.6. Separation of bIgG from the BSA containing protein mixture T-ABI expanded bed was used to separate bIgG from the protein mixture (2 mg/ml bIgG and 10 mg/ml BSA) as mentioned above. The loading and elution pH were set as pH 8.0 and pH 3.5, respectively. Four expansion factors (1.8, 2.0, 2.2 and 2.4) were tested. The recoveries and purities were compared in Fig. 9. It was found that the recoveries and purities hardly changed for the expanded factors tested and remained around 79% and 78%, respectively. The highest recovery and purity were obtained at the expansion factor of 2.0 with 80.6% and 78.2%. Therefore, expansion factor 2.0 was chosen as the best operation condition for EBA, which was consistent with the results of dynamic binding capacity. The effects of loading volume were investigated further for EBA applications. As shown in Fig. 10, the recoveries of bIgG decreased (81.8–66.4%) and the purities increased (75.5–84.1%) with the increase of loading volume from 1-fold to 4-fold settled bed volume. The reason might be that there was competitive adsorption
Fig. 8. Breakthrough curves of bIgG with T-ABI in expanded bed at different flow velocities. Solid line, 711 cm/h; dash line, 889 cm/h; dot line, 1050 cm/h; dash dot line, 1203 cm/h.
Fig. 9. Recovery and purity of bIgG with T-ABI expanded bed at different expansion factors. Breakthrough with 20 mM phosphate buffer (pH 8.0); elution with 20 mM acetate buffer (pH 3.5); CIP with 0.1 M NaOH. The operation velocities were 711, 889, 1050 and 1203 cm/h, respectively.
between bIgG and BSA on T-ABI, and bIgG showed stronger binding than BSA. When the sample loading was low, the adsorption sites on T-ABI were enough for both bIgG and BSA and the competitive adsorption was not apparent, which led to adsorption for both proteins. So the recovery of bIgG was high, but the purity was relatively low. When the sample loading was high, the competitive adsorption would become stronger and some BSA would be replaced by bIgG, which resulted in the increase of bIgG purity but the decrease of bIgG recovery due to the overload and breakthrough of bIgG. In view of both purity and recovery, 2-fold settled bed volume was chosen as the best loading condition, resulting in the hIgG purity of 78.2% and recovery of 80.6%. 3.7. Separation of bIgG from bovine whey Finally, T-ABI expanded bed was challenged to separate bIgG directly from crude bovine whey. The HPLC analysis indicated that bIgG concentration in whey was about 0.6 mg/ml, which accounted for 4.7% of total protein. Due to low proportion of bIgG and many
Fig. 10. Recovery and purity of bIgG with T-ABI expanded bed at different loading volumes. Breakthrough with 20 mM phosphate buffer (pH 8.0); elution with 20 mM acetate buffer (pH 3.5); CIP with 0.1 M NaOH. The operation velocity was 889 cm/h.
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3.5. High purity and recovery of bIgG could be obtained, 78.2% and 80.6% for the protein mixture, 90.6% and 78.2% for bovine whey, respectively. The results indicated that T-ABI could be used for the separation of bIgG from the complex feedstock with high separation efficiency, and hydrophobic charged-induction EBA with ABI as the functional ligand would be a promising new technology for antibody purification. Acknowledgment This work was financially supported by the National Natural Science Foundation of China and the International Science & Technology Cooperation Program of China. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma.2015.10. 055. References Fig. 11. SDS-PAGE analysis of fractions collected during the separation of bIgG from bovine whey with T-ABI expanded bed. Lane 1, protein marker; 2, loading sample; 3, breakthrough; 4, elution.
impurities in whey, Q10% of bIgG decreased to about 1.8 mg/ml resin (as shown in Fig. S3 in supporting information). Under the optimized operation conditions (loading at pH 8.0 with 40 ml bovine whey, expansion factor of 2.0, elution at pH 3.5), bIgG was separated successfully with high purity (90.6%) and suitable recovery (78.2%). The analysis of SDS-PAGE of collected fractions is shown in Fig. 11, and SEC-HPLC results are shown in Fig. S4 (supporting information). It could be found that main impurities in whey were some low-molecular-weight proteins. During the EBA separation, most of impurities could flow through the column and high purity of bIgG could be obtained with one-step EBA process. The purification factor reached about 19.3. Compared with bIgG separation from the protein mixture in Section 3.6, the purity of bIgG from bovine whey was improved due to low BSA concentration in whey. As shown in Fig. 11, two weak bands around 60–80 kDa appeared between BSA and bIgG in SDS-PAGE, which would be lactoperoxidase and lactoferrin as main impurities [19,31]. The results demonstrated that new EBA process with ABI as the functional hydrophobic chargedinduction ligand would be a promising new technology for IgG purification. 4. Conclusions HCIC ligands MEP and ABI were coupled onto the agarose beads containing tungsten carbide to prepare two EBA resins, T-MEP and T-ABI. The static adsorption and dynamic binding behaviors of bIgG were determined. It was found that T-MEP and T-ABI had similar static adsorption behaviors, including high adsorption of bIgG, pH dependence and salt tolerance. However, T-ABI showed higher dynamic binding capacity in packed bed than T-MEP, which might be due to the relatively strong hydrophobic property of ABI ligand. Further studies in expanded bed indicated that T-ABI could be used for high operation velocity and a stable bed could be maintained even for high flow velocity. Moreover, dynamic binding capacities of bIgG in expanded bed were similar to that in packed bed. Finally, EBA with T-ABI was optimized to separate bIgG from the BSA containing feedstock (2 mg/ml bIgG and 10 mg/ml BSA) and crude bovine whey. The suitable operation conditions were set as the expansion factor of 2.0, loading at pH 8.0 and elution at pH
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Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
5-Aminobenzimidazole as new hydrophobic charge-induction ligand for expanded bed adsorption of bovine IgG Wei Shi a , Dong-Qiang Lin a,∗ , Hong-Fei Tong a , Jun-Xian Yun b , Shan-Jing Yao a a Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China b State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China
a r t i c l e
i n f o
Article history: Received 6 August 2015 Received in revised form 24 September 2015 Accepted 16 October 2015 Available online 24 October 2015 Keywords: Expanded bed adsorption Hydrophobic charge-induction chromatography 5-Aminobenzimidazole Bovine whey IgG
a b s t r a c t Expanded bed adsorption (EBA) can capture target proteins directly from unclarified feedstock without prior solid–liquid separation. Hydrophobic charge-induction chromatography (HCIC) is a promising technology for biomolecule separation with high capacity, good selectivity and relatively low cost without the pretreatment of dilution or salt addition. In this work, EBA and HCIC were combined to develop a new separation technology, hydrophobic charge-induction EBA. Two HCIC ligands, 4-mercapto-ethyl-pyridine (MEP) and 5-aminobenzimidazole (ABI), were coupled onto agarose beads containing tungsten carbide to prepare the resins for EBA, named T-MEP and T-ABI, respectively. The static adsorption and dynamic binding behaviors of bovine IgG (bIgG) were investigated. Two resins had similar saturated adsorption capacities and salt-tolerant properties, but T-ABI showed higher dynamic binding capacity than T-MEP, indicating that ABI ligand was more suitable for EBA. The performances in expanded bed were verified. With the protein mixture (2 mg/ml bIgG and 10 mg/ml bovine serum albumin) as the model feedstock, the effects of loading and elution pH, expansion factor and loading volume on the separation performance of bIgG were evaluated. Finally, T-ABI EBA was used to separate bIgG directly from bovine whey with optimized operation conditions. The purity and recovery of bIgG reached 90.6% and 78.2%, respectively. The purification factor was about 19.3. The results demonstrated that the combination of HCIC and EBA would be a potential platform for antibody capture with less feedstock pretreatments, high efficiency and relatively low cost. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Expanded bed adsorption (EBA) was developed in 1990s as an innovative technology that allows capturing target proteins directly from unclarified feedstock, e.g., culture suspensions and cell homogenates [1–3], which combines solid–liquid separation with an adsorption step in a single-unit operation [4,5]. When the mobile phase is pumped upward through the column, the resin beads with an appropriate distribution of size/density can form a perfectly classified fluidized bed with low back-mixing. Cell or cell debris can pass through the voidage between resin beads without blocking the column and the target can be adsorbed by the resin. Obviously, EBA technology can reduce operational time and increased overall yield with less requirements for capital investment and consumables. In general, specially designed resins are
∗ Corresponding author. E-mail address: [email protected] (D.-Q. Lin). http://dx.doi.org/10.1016/j.chroma.2015.10.055 0021-9673/© 2015 Elsevier B.V. All rights reserved.
critical for EBA operation, which have a significant influence on the feasibility and the overall performance of the process. During the past 20 years, series of matrices have been developed for EBA. For example, Streamline and Streamline Direct are two series of the well-known commercial resins for EBA developed by GE Healthcare (Uppsala, Sweden), which are based on cross-linked agarose containing a crystalline quartz core or stainless steel power as the densifier. These resins show some applications for protein separation, but new series of resins are still needed to improve the performance of EBA. The functional ligands coupled on the resin beads are an important factor to determine the adsorption efficiency of EBA process. The most common applications for EBA are ion-exchange and hydrophobic interaction resins. However, due to the complexity of biomolecular structure, the separation selectivity of these two kinds of binding modes is normally limited. In addition, the conductivity of the feedstock should be considered and some pretreatments have to be done, such as the dilution for ion exchange and the addition of salt for hydrophobic interaction separations,
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Fig. 1. Preparation scheme of two HCIC resins T-MEP and T-ABI.
which certainly increase the cost and process time. Hydrophobic charge-induction chromatography (HCIC) with dual-mode ionizable ligands is new protein separation method [6], which often shows high capacity at a wide range of salt concentration. HCIC ligands, such as 4-mercapto-ethyl-pyridine (MEP) with the pKa of 4.8, show a typical pH-dependent property, from non-charge to positive charges with the decrease of pH. The target protein can be adsorbed on the uncharged ligand at neutral pH by the hydrophobic interactions, and desorbed by the electrostatic repulsion between target protein and the charged ligand at acidic condition. HCIC has been applied to the purification of antibodies and other proteins, which showed a potential alternative to Protein A chromatography [7–10]. Xia et al. [11] coupled three HCIC ligands, MEP, 2-mercapto1-methyl-imidazole (MMI) and 2-mercapto-benzimidazole (MBI) onto the macroporous cellulose-tungsten carbide composite beads, and used for EBA separation of immunoglobulin Y (IgY) from egg yolk. The results demonstrated that HCIC in expanded bed mode is a potential technology for antibody separation both in high efficiency and high productivity. However, due to weak hydrophobic interactions between target protein and the ligands used, the dynamic binding capacities were relatively low, especially for high operation velocity for EBA. Therefore, new HCIC ligands should be developed to enhance the binding forces and improve the applications for EBA processes. In the present work, the tungsten-carbide-densified agarose beads were used as the matrix for EBA. The agarose beads containing tungsten carbide as the densifier have relatively high density (2.8–3.2 g/ml) compared to the beads with quartz glass (Streamline series) and steel core (Streamline Direct series), which can be used for high superficial velocity and lead to high production. Two HCIC ligands, MEP and 5-aminobenzimidazole (ABI), were coupled onto the matrix to prepared new resins for EBA. The former represents the typical HCIC ligand, and the latter is newly-designed ligand based on the molecular simulation [12]. The structures of MEP and ABI are shown in Fig. 1. Compared with MEP with a pyridine ring, ABI had two cyclic structures including one benzene ring and one imidazole ring, which might improve the hydrophobic binding with target protein. The pKa of ABI is 6.5, resulting in milder pH condition for the charge-induction effect and protein elution. In addition, the results of molecular simulation showed that ABI could bind onto the consensus binding site of Fc domain of IgG molecule in a favorable way [12], so ABI was considered as a new HCIC
ligand for antibody purification. Therefore, two ligands, MEP and ABI, would be evaluated and compared for EBA process in this work. Firstly, the adsorption behaviors of bovine IgG (bIgG) on two resins would be investigated with batch and frontal adsorption experiments. The effects of pH and salt on the static and dynamic binding capacity would be focused. Then, the separation of bIgG from the bovine serum albumin (BSA)-containing feedstock would be investigated, and the bed expansion and liquid mixing in expanded bed were evaluated. Finally, EBA process was developed to separate bIgG from bovine whey. The potential of the application of new HCIC resins for antibody purification with EBA process would be discussed. 2. Materials and methods 2.1. Materials ABI and MEP were purchased from J&K technology Co., Ltd. (China). Crosslinked agarose beads containing tungsten carbide were prepared with similar methods as published in our previous work [11,13]. Bovine serum ␥-globulin (bovine IgG, electrophoresis purity 99.0%) was purchased from Merck KGaA (Darmstadt, Germany). BSA (67 kDa) was purchased from Sigma (Milwaukee, WI, USA). Bovine milk was obtained from local resource. All other chemical materials were of analytical grade and were purchased commercially. 2.2. Preparation of resins The preparation procedure was similar to that published by Gao et al. [13] and Xia et al. [11]. The matrices were activated with allyl bromide at the basic condition and brominated by Nbromosuccinimide (NBS), then coupled with HCIC ligands, MEP or ABI, under proper conditions. Typically, 10 g drained agarose beads were mixed with 4 ml allyl bromide and 5 g NaOH in 11 ml 20% dimethyl sufoxide (DMSO), and the mixture was continuously agitated at 180 rpm and 50 ◦ C for 24 h. The allyl-activated matrices were washed with deionized water and mixed with 10 ml H2 O, 10 ml acetone, and 3 molar excess of NBS over the allyl groups and agitated at 180 rpm and 30 ◦ C for 1 h. Then, the brominated matrices were mixed with 3 molar excess of MEP or ABI over the allyl groups in 10 ml 1 M Na2 CO3 (pH ∼11.7), and agitated at 180 rpm
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and 30 ◦ C for 12 h. Finally the beads were washed with 0.1 M HCl, 0.1 M NaOH and deionized water, and the resins were stored in 20% (v/v) ethanol. Two kinds of HCIC resins were prepared, namely T-MEP and T-ABI, respectively. 2.3. Adsorption equilibrium experiments The static adsorption behaviors of bIgG on two resins were measured by batch adsorption equilibrium experiments with the procedure as the references [14,15]. 20 mM sodium acetate buffer (pH 5.0), 20 mM phosphate buffer (pH 6.0, pH 7.0 and pH 8.0) and 20 mM glycine buffer (pH 9.0 and pH 10.0) were used as the liquid phases for different pHs. After the adsorption reached equilibrium, the protein concentration in the supernatant was determined at 280 nm with One Drop Spectrophotometer (Nanjing WuYi Technology Co., Ltd., Nanjing, China). The adsorption isotherm was fitted with the Langmuir equation, and the saturated adsorption capacity (Qm ) and the dissociation constant (Kd ) were obtained. Ammonium sulfate and sodium chloride were also added into the buffer (0, 0.2, 0.4, 0.6 and 0.8 M), respectively, and used as the liquid phase to investigate the influence of salt concentration on the adsorption of bIgG on two resins. 2.4. Evaluation with packed bed 5-mm I.D. column (Tricorn 5/100, GE Healthcare, Uppsala, Sweden) was packed with 2.0 ml resins (about 10 cm settled bed height). The dynamic binding capacity was investigated through frontal adsorption experiments. The column was equilibrated with the equilibrium buffer. Then, bIgG solution (2 mg/ml) was loaded into the column at the superficial linear velocity of 200, 400 or 600 cm/h. The protein concentration in the effluent was monitored by absorbance at 280 nm with a UV monitor LC3000 (Beijing Chuangxintongheng Science & Technology Co., Ltd., Beijing, China). The dynamic binding capacity at 10% breakthrough (Q10% ) was calculated as described by Griffith et al. [16]. The separation of bIgG from BSA containing feedstock was evaluated with the same column and the ÄKTA explorer 100 system (GE Healthcare, Uppsala, Sweden). Loading and elution pH were screened. 20 mM phosphate buffer (pH 7.0 and pH 8.0) and 20 mM glycine buffer (pH 9.0) were used as the loading buffer, and 20 mM acetate buffer (pH 3.0, 3.5, 4.0 and 4.5) were used as the elution buffer. The protein mixture containing 2 mg/ml bIgG and 10 mg/ml BSA was used as the loading feedstock. The flow rate was 0.5 ml/min. The column was equilibrated with the loading buffer and then 2 ml protein mixture (corresponding to 1-fold column volume) was loaded. After sample loading, the column was washed by the loading buffer and then bIgG was eluted with the acetate buffer. Finally, the column was regenerated with 0.1 M NaOH and re-equilibrated with the loading buffer for the next test. The chromatographic run was monitored on-line at 280 nm. The fractions were collected and analyzed with the SEC-HPLC and SDS-PAGE. 2.5. Characteristics in expanded bed 1 cm-diameter home-made column was used, and 13.5 ml resins (about 19 cm settled bed height, H0 ) were packed into the column. All experiments were performed at room temperature and the proper column vertical alignment was assured. The bed was expanded with 20 mM sodium phosphate buffer (pH 8.0) and the equilibrium state was reached after about 20–30 min of expansion and a stable bed height was obtained. The expanded bed height (H) was measured three times for one flow rate, and then the mean value was used to calculate the expansion factor (H/H0 ). Six expansion factors (1.6, 1.8, 2.0, 2.2, 2.4 and 2.6) were tested.
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Liquid mixing in expanded bed was evaluated by the measurement and analysis of residence time distribution (RTD). 0.5 ml acetone solution (10%, w/w) was used as the tracer. The response signal at the outlet was detected by the UV detector (LC3000, Beijing Chuangxintongheng Science & Technology Co., Ltd., Beijing, China). The RTD test for each sampling port was repeated 3–4 times. The axial dispersion coefficient (Dax ) was calculated based on the RTD curves [17,18]. 2.6. Frontal adsorption experiments in expanded bed The dynamic binding capacity of bIgG in expanded bed was determined by frontal adsorption experiments. 1 cm-diameter column and 13.5 ml resins (about 19 cm settled bed height) were used. The bed was firstly expanded with 20 mM sodium phosphate buffer (pH 8.0) and the equilibrium state was reached after 20–30 min. Four expansion factors (1.8, 2.0, 2.2 and 2.4) were tested. 2 mg/ml bIgG solution was then pumped into the column. The protein breakthrough at outlet was detected at 280 nm with LC3000. The dynamic binding capacity at 10% breakthrough was calculated as mentioned above. 2.7. Separation of bIgG from protein mixture with expanded bed 1 cm-diameter column and 13.5 ml resins (about 19 cm settled bed height) were used. The protein mixture containing 2 mg/ml bIgG and 10 mg/ml BSA was used as the loading feedstock as mentioned above. The influences of expansion factor (1.8, 2.0, 2.2 and 2.4) and loading volume (1, 2, 3 and 4 folds of settled bed volume) were investigated with the expanded bed system as mentioned above. The bed was expanded and equilibrated with 20 mM sodium phosphate buffer (pH 8.0) until a stable bed height was obtained. The protein mixture was loaded into the column. Then, the column was washed with the loading buffer to remove unbound proteins. After that, bIgG was eluted with 20 mM acetate buffer (pH 3.5). Finally, the column was regenerated with 0.1 M NaOH and reequilibrated with the loading buffer. The fractions were collected and analyzed with the SEC-HPLC and SDS-PAGE. The EBA process was repeated 3 times, and the average recovery and purity of bIgG were obtained. 2.8. Separation of bIgG from bovine whey with expanded bed Bovine whey was prepared as published previously [19]. The main processes were as follows. Bovine milk was centrifuged for 30 min at 5000 rpm to remove fat, and then adjusted to pH 4.7 by the addition of 5 M HCl. Afterwards, casein was precipitated from bovine milk by stirring at 35 ◦ C for 50 min, and bovine whey was obtained by centrifugation. The pH was adjusted to 8.0 with 2 M NaOH for the further EBA tests. The concentration and purity of bIgG in whey were analyzed with SEC-HPLC. Same expanded bed as mentioned above was used for bIgG separation. For bIgG separation from bovine whey, the bed with 13.5 ml T-ABI resins was expanded to 2.0 expansion factor with 20 mM sodium phosphate buffer (pH 8.0). 40 ml bovine whey was loaded. After sample loading, the column was washed with the equilibrium buffer. Then bIgG was eluted with 20 mM acetate buffer (pH 3.5). The column was regenerated with 0.1 M NaOH and re-equilibrated with the equilibrium buffer. The fractions were collected during the process and analyzed with the SEC-HPLC and SDS-PAGE to determine the recovery and purity of bIgG. 2.9. SEC-HPLC analysis The analytical SEC-HPLC was performed on LC3000 system (Beijing Chuangxintongheng Science & Technology Co., Ltd.,
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Beijing, China) with the TSK G3000SWXL column (7.8 mm × 30.0 cm, TOSOH, Japan). 0.1 M sodium phosphate buffer (pH 6.7, containing 0.1 M Na2 SO4 ) was used as the mobile phase. The buffer was filtrated with 0.22 m membrane and degassed before use. The flow rate was 0.5 ml/min. The purity of bIgG was defined as the percentage of the peak area of bIgG monomer to the total integrated peak areas. The recovery of bIgG was calculated as the percentage of bIgG monomer in the elution fraction to that in the loading sample during the separation process. 2.10. SDS-PAGE analysis The sample and fractions collected during the separation process were analyzed by 12% non-reducing SDS-PAGE. Protein migration was performed under 180 V for 50 min. The gel was stained with Coomassie Blue R-250 and destained. The Gel Doc 2000 imaging system (Bio-Rad, Hercules, CA, USA) was used to image the protein gel. 3. Results and discussion 3.1. Preparation of T-MEP and T-ABI resins Two HCIC resins, T-MEP and T-ABI, with tungsten carbideagarose composite beads as the matrix and MEP/ABI as the functional ligand were prepared with the route as shown in Fig. 1. The agarose beads were activated with allyl bromide firstly, and the density of allyl group on the activated bead was about 125 mol/ml resin. After brominated by NBS, the ligand coupling reaction was conducted under alkali condition as published previously [11,13]. The ligand densities of MEP and ABI on the resins prepared were 86 and 76 mol/ml resin, respectively. The results indicated that the preparation procedure was suitable, and the coupling efficiencies of two ligands were similar. 3.2. Adsorption equilibrium of bIgG on T-MEP and T-ABI resins The pH-dependent and salt-tolerant adsorption behaviors are typical characteristics of HCIC resins. Thus, it is important to investigate the influences of pH and salt concentration on the adsorption capacity of two new resins firstly. The batch experiments were adopted to study the static adsorption behaviors of bIgG. The adsorption isotherms of bIgG with T-MEP and T-ABI at different pH values and salt concentrations were determined. As shown in Fig. 2 and Table 1, pH had a significant influence on the adsorption of bIgG on both resins. For T-MEP, the highest adsorption capacities were found at pH 9.0 with the Qm of 86.45 mg/ml resin, and Kd was 0.30 mg/ml. The Qm values at pH 7.0 and 8.0 were comparable, i.e. 75.71 and 81.68 mg/ml resin, respectively. When the pH decreased to 5.0, the adsorption capacity of bIgG decreased remarkably. For T-ABI, the results were similar to Table 1 Equilibrium parameters Qm and Kd of bIgG adsorption with T-MEP and T-ABI at various pHs. pH
5.0 6.0 7.0 8.0 9.0 10.0
T-MEP
T-ABI
Qm (mg/ml resin)
Kd (mg/ml)
Qm (mg/ml resin)
Kd (mg/ml)
40.72 70.72 75.71 81.68 86.45 76.90
0.87 0.57 0.49 0.39 0.30 0.38
37.78 65.23 66.55 74.68 73.54 60.79
0.52 0.31 0.25 0.23 0.25 0.36
Fig. 2. The adsorption isotherms of bIgG with T-MEP (a) and T-ABI (b) at different pH values. (䊉) pH 5.0; () pH 6.0; () pH 7.0; () pH 8.0; () pH 9.0; () pH 10.0.
that of T-MEP. High adsorption capacities and low Kd values were also found at pH 7.0, 8.0 and 9.0. In addition, the adsorption capacity at pH 5.0 was quite low as well. Compared with T-MEP, T-ABI showed slightly lower adsorption capacities and Kd values. This might be due to relatively lower ligand density of T-ABI but stronger hydrophobic interactions of ABI ligand. For both resins, pH 7.0–9.0 would be suitable for bIgG adsorption and the acidic pH could be used for the elution. The effects of (NH4 )2 SO4 and NaCl addition on the adsorption equilibrium of bIgG with T-MEP and T-ABI at pH 8.0 are shown in Figs. S1 and S2 (supporting information), and the equilibrium parameters Qm and Kd are listed in Tables S1and S2 (supporting information). It was found that some adsorption isotherms did not reach the plateaus, especially for high salt concentration, so the fitted Qm values would be not suitable to describe adsorption ability. As suggested by Sun and coworkers [20–23], the adsorbed protein density (Qc ) at an equilibrium liquid-phase concentration calculated by Langmuir equation was introduced. The Qc values at the equilibrium liquid-phase concentration of 5 mg/ml were calculated and compared in Fig. 3. It could be found that two resins had similar “U” shape trends with the increase of salt concentration. Qc of bIgG declined slightly when salt concentration increased from 0 to 0.2 M. Then the adsorption capacity increased gradually with the increase of salt concentration from 0.2 to 0.8 M. For high salt concentration, Qc values were even higher than that without salt addition.
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Fig. 4. Comparison of Q10% with T-MEP and T-ABI in packed bed at different flow velocities.
Fig. 3. The Qc s of bIgG with T-MEP (a) and T-ABI (b) at pH 8.0 and the equilibrium liquid-phase concentration of 5 mg/ml as a function of different ammonium sulfate and sodium chloride concentrations.
The decline of the adsorption capacity for both resins at 0.2 M (NH4 )2 SO4 or NaCl might be due to the impact on the hydrogen bonds between HCIC ligand and bIgG under low salt concentration. When salt concentration increased continuously, salting-out effect would lead to the exposure of hydrophobic area on the surface of protein and improve the hydrophobic interactions between HCIC ligand and bIgG, resulting in the increase of adsorption capacity. In general, the adsorption capacities of T-MEP and T-ABI did not change significantly with the change of salt concentration, which indicated both resins had good property of salt tolerance.
ligand could provide stronger hydrophobic interactions with target IgG molecule, which certainly fasten the mass transfer in the pores and improve the rate of protein adsorption. Therefore, compared with T-MEP, T-ABI was more suitable for application for EBA with high flow velocity, and would be further studied for the applications of protein separation and EBA tests. T-ABI resin was challenged to purify bIgG from the protein mixture of bIgG and BSA with packed bed. The feedstock contained 2 mg/ml bIgG and 10 mg/ml BSA. The loading and elution pH were screened to optimize the separation efficiency. The chromatographic results at different loading pH conditions are shown in Fig. 5. With the sample loading at pH 7.0 and elution at pH 4.0, the recovery and purity of bIgG were 70.2% and 73.4%, respectively. When the loading pH increased to pH 8.0, the purity of bIgG decreased slightly (72.4%), but the recovery increased obviously (82.2%). At pH 9.0, bIgG had the highest purity of 76.8%, while the recovery decreased to 74.4%. Considering the recovery and purity together, pH 8.0 was chosen as the suitable loading pH for following separation experiments. The elution pH was optimized further, and the results are shown in Fig. 6. It was found that the elution peak heights increased with the decrease of elution pH from 4.5 to 3.0, indicating that the elution of bIgG was enhanced apparently at acidic condition. The recoveries of bIgG were 61.9%, 82.2%, 92.3% and 94.3% for the elution pH of 4.5, 4.0, 3.5 and 3.0, respectively. Highest purity of bIgG (90.3%) was found at the elution pH 4.5, while a relatively low purity of bIgG (67.6–72.4%) was found for the elution of pH 3.0–4.0. Considering the purity and recovery together, the elution at pH 3.5 would be suitable for bIgG separation from the BSA containing feedstock. 3.4. Expansion characteristics and liquid mixing in expanded bed
3.3. Dynamic binding capacity and protein separation in packed bed Dynamic binding capacities of bIgG with two resins at different flow velocities were evaluated by the frontal adsorption experiments in packed bed. The dynamic binding capacities at 10% breakthrough, Q10% , are compared in Fig. 4. Although the static adsorption capacities of T-ABI and T-MEP were similar, TABI had higher Q10% than T-MEP. With the increase of flow velocity from 200 to 600 cm/h, Q10% of T-ABI decreased from 15.15 to 6.74 and 5.21 mg/ml resin. For T-MEP, Q10% was only 5.65 mg/ml resin at 200 cm/h and decreased to 3.60 mg/ml resin at 400 cm/h and 2.53 mg/ml resin at 600 cm/h. The results indicated further that ABI
Expansion characteristic is one of the most important factors for EBA process, which affects greatly the adsorption performance. Normally the expansion factor is controlled at the range of 1.5–3.0 [24–28]. When the expansion factor is too low, the bed could not be expanded adequately. The excessive fluidization and the loss of small resin beads would be found when the expansion factor is too high. Therefore, based on the tungsten carbide-agarose composite beads used in the present work, the expansion factors of 1.6–2.6 were tested. The results are shown in Fig. 7. Due to high wet density of T-ABI resin with tungsten carbide as the densifier, high operation velocity (550–1355 cm/h) was needed for the expansion factors of 1.6–2.6. For the normal expansion factor of 2.0 for EBA process,
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Fig. 5. Chromatograms of bIgG separation at different loading pHs with T-ABI packed bed. Breakthrough (BT) with 20 mM phosphate buffer (pH 7.0, pH 8.0) and 20 mM glycine buffer (pH 9.0); elution with 20 mM acetate buffer (pH 4.0); CIP with 0.1 M NaOH. The flow rate was 0.5 ml/min.
the operation velocity was 889 cm/h with T-ABI, which was much higher than some commercial EBA resins, such as 264 cm/h for Streamline series and 517 cm/h for Streamline Direct series [29,30]. Liquid mixing in the expanded bed is an important property for evaluating the bed stability and protein adsorption. The liquid mixing and bed stability were evaluated with RTD tests at different expansion factors. The theoretical plate number and Dax were calculated. Small Dax means lower back mixing and a more stable bed. Dax as the function of flow velocity is shown in Fig. 7. It was found that the Dax increased with the increase of flow velocity, and all Dax values kept at a level of 10−5 m2 /s. Even for high flow velocity of about 1300 cm/h at the expansion factor of 2.6, the bed was still quite stable to meet the requirement of expanded bed operation.
3.5. Dynamic binding capacity in expanded bed The dynamic binding behaviors of bIgG with T-ABI in expanded bed at varying flow velocities were evaluated by the front adsorption experiments. As shown in Fig. 8, the breakthrough curves at flow velocities of 711, 889, 1050 and 1203 cm/h (corresponding to expansion factors 1.8, 2.0, 2.2 and 2.4, respectively) were quite similar. It was found that the Q10% s at varying flow velocities were close to each other (4.53 mg/ml resin for EF 1.8, 5.41 mg/ml resin for EF 2.0, 5.49 mg/ml resin for EF 2.2 and 5.10 mg/ml resin for EF 2.4), indicating that the expanded bed was quite stable. Q10% values in expanded bed were also near that in packed bed at 600 cm/h, which indicated that ABI ligand had a strong ability to capture bIgG
Fig. 6. Chromatograms of bIgG separation at different elution pHs with T-ABI packed bed. Breakthrough (BT) with 20 mM phosphate buffer (pH 8.0); elution with 20 mM acetate buffer (pH 3.0, 3.5, 4.0 and 4.5); CIP with 0.1 M NaOH. The flow rate was 0.5 ml/min.
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Fig. 7. Expansion factor and Dax as the function of flow velocity in expanded bed.
and a good protein adsorption could be maintained even for high flow velocity as 1000 cm/h. Compared different flow velocities, the operations at 889 cm/h (expansion factor of 2.0) and 1050 cm/h (expansion factor of 2.2) reached the highest dynamic binding capacities, which would be most suitable for EBA applications. 3.6. Separation of bIgG from the BSA containing protein mixture T-ABI expanded bed was used to separate bIgG from the protein mixture (2 mg/ml bIgG and 10 mg/ml BSA) as mentioned above. The loading and elution pH were set as pH 8.0 and pH 3.5, respectively. Four expansion factors (1.8, 2.0, 2.2 and 2.4) were tested. The recoveries and purities were compared in Fig. 9. It was found that the recoveries and purities hardly changed for the expanded factors tested and remained around 79% and 78%, respectively. The highest recovery and purity were obtained at the expansion factor of 2.0 with 80.6% and 78.2%. Therefore, expansion factor 2.0 was chosen as the best operation condition for EBA, which was consistent with the results of dynamic binding capacity. The effects of loading volume were investigated further for EBA applications. As shown in Fig. 10, the recoveries of bIgG decreased (81.8–66.4%) and the purities increased (75.5–84.1%) with the increase of loading volume from 1-fold to 4-fold settled bed volume. The reason might be that there was competitive adsorption
Fig. 8. Breakthrough curves of bIgG with T-ABI in expanded bed at different flow velocities. Solid line, 711 cm/h; dash line, 889 cm/h; dot line, 1050 cm/h; dash dot line, 1203 cm/h.
Fig. 9. Recovery and purity of bIgG with T-ABI expanded bed at different expansion factors. Breakthrough with 20 mM phosphate buffer (pH 8.0); elution with 20 mM acetate buffer (pH 3.5); CIP with 0.1 M NaOH. The operation velocities were 711, 889, 1050 and 1203 cm/h, respectively.
between bIgG and BSA on T-ABI, and bIgG showed stronger binding than BSA. When the sample loading was low, the adsorption sites on T-ABI were enough for both bIgG and BSA and the competitive adsorption was not apparent, which led to adsorption for both proteins. So the recovery of bIgG was high, but the purity was relatively low. When the sample loading was high, the competitive adsorption would become stronger and some BSA would be replaced by bIgG, which resulted in the increase of bIgG purity but the decrease of bIgG recovery due to the overload and breakthrough of bIgG. In view of both purity and recovery, 2-fold settled bed volume was chosen as the best loading condition, resulting in the hIgG purity of 78.2% and recovery of 80.6%. 3.7. Separation of bIgG from bovine whey Finally, T-ABI expanded bed was challenged to separate bIgG directly from crude bovine whey. The HPLC analysis indicated that bIgG concentration in whey was about 0.6 mg/ml, which accounted for 4.7% of total protein. Due to low proportion of bIgG and many
Fig. 10. Recovery and purity of bIgG with T-ABI expanded bed at different loading volumes. Breakthrough with 20 mM phosphate buffer (pH 8.0); elution with 20 mM acetate buffer (pH 3.5); CIP with 0.1 M NaOH. The operation velocity was 889 cm/h.
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3.5. High purity and recovery of bIgG could be obtained, 78.2% and 80.6% for the protein mixture, 90.6% and 78.2% for bovine whey, respectively. The results indicated that T-ABI could be used for the separation of bIgG from the complex feedstock with high separation efficiency, and hydrophobic charged-induction EBA with ABI as the functional ligand would be a promising new technology for antibody purification. Acknowledgment This work was financially supported by the National Natural Science Foundation of China and the International Science & Technology Cooperation Program of China. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma.2015.10. 055. References Fig. 11. SDS-PAGE analysis of fractions collected during the separation of bIgG from bovine whey with T-ABI expanded bed. Lane 1, protein marker; 2, loading sample; 3, breakthrough; 4, elution.
impurities in whey, Q10% of bIgG decreased to about 1.8 mg/ml resin (as shown in Fig. S3 in supporting information). Under the optimized operation conditions (loading at pH 8.0 with 40 ml bovine whey, expansion factor of 2.0, elution at pH 3.5), bIgG was separated successfully with high purity (90.6%) and suitable recovery (78.2%). The analysis of SDS-PAGE of collected fractions is shown in Fig. 11, and SEC-HPLC results are shown in Fig. S4 (supporting information). It could be found that main impurities in whey were some low-molecular-weight proteins. During the EBA separation, most of impurities could flow through the column and high purity of bIgG could be obtained with one-step EBA process. The purification factor reached about 19.3. Compared with bIgG separation from the protein mixture in Section 3.6, the purity of bIgG from bovine whey was improved due to low BSA concentration in whey. As shown in Fig. 11, two weak bands around 60–80 kDa appeared between BSA and bIgG in SDS-PAGE, which would be lactoperoxidase and lactoferrin as main impurities [19,31]. The results demonstrated that new EBA process with ABI as the functional hydrophobic chargedinduction ligand would be a promising new technology for IgG purification. 4. Conclusions HCIC ligands MEP and ABI were coupled onto the agarose beads containing tungsten carbide to prepare two EBA resins, T-MEP and T-ABI. The static adsorption and dynamic binding behaviors of bIgG were determined. It was found that T-MEP and T-ABI had similar static adsorption behaviors, including high adsorption of bIgG, pH dependence and salt tolerance. However, T-ABI showed higher dynamic binding capacity in packed bed than T-MEP, which might be due to the relatively strong hydrophobic property of ABI ligand. Further studies in expanded bed indicated that T-ABI could be used for high operation velocity and a stable bed could be maintained even for high flow velocity. Moreover, dynamic binding capacities of bIgG in expanded bed were similar to that in packed bed. Finally, EBA with T-ABI was optimized to separate bIgG from the BSA containing feedstock (2 mg/ml bIgG and 10 mg/ml BSA) and crude bovine whey. The suitable operation conditions were set as the expansion factor of 2.0, loading at pH 8.0 and elution at pH
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