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Evaluation on adsorption selectivity of immunoglobulin G with 2-mercapto-1-methyl-imidazole-based hydrophobic charge-induction resins
Accepted Manuscript Title: Evaluation on adsorption selectivity of immunoglobulin G with 2-mercapto-1-methyl-imidazole-based hydrophobic charge-induction resins Author: Hui-Li Lu Dong-Qiang Lin Qi-Lei Zhang Shan-Jing Yao PII: DOI: Reference:
S1369-703X(16)30338-2 http://dx.doi.org/doi:10.1016/j.bej.2016.12.005 BEJ 6612
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
5-9-2016 1-12-2016 3-12-2016
Please cite this article as: Hui-Li Lu, Dong-Qiang Lin, Qi-Lei Zhang, Shan-Jing Yao, Evaluation on adsorption selectivity of immunoglobulin G with 2-mercapto-1-methylimidazole-based hydrophobic charge-induction resins, Biochemical Engineering Journal http://dx.doi.org/10.1016/j.bej.2016.12.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Evaluation on adsorption selectivity of immunoglobulin G with 2-mercapto-1-methyl-imidazole-based hydrophobic charge-induction resins
Hui-Li Lu, Dong-Qiang Lin*, Qi-Lei Zhang, Shan-Jing Yao*
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
Corresponding author: Prof. Dong-Qiang Lin and Prof. Shan-Jing Yao College of Chemical and Biological Engineering Zhejiang University Hangzhou 310027 China Fax: +86-571-87951982 E-mail: [email protected]; [email protected]
1
Research highlights ► 2-mercapto-1-methyl-imidazole-based hydrophobic charge-induction resins were evaluated. ► Competitive adsorption behaviors between immunoglobulin G and bovine serum albumin were investigated ► Resins with moderate pore size and high ligand density exhibited the best adsorption selectivity. ► Immunoglobulin G was preferentially adsorbed. ► Immunoglobulin G was separated from protein mixtures effectively.
2
ABSTRACT
Hydrophobic
charge-induction
chromatography
(HCIC)
with
2-mercapto-1-methyl-imidazole (MMI) ligands shows promising results in antibody purification. In this study, competitive adsorption processes between bovine immunoglobulin G (IgG) and bovine serum albumin (BSA) were investigated with MMI resins. Adsorption isotherms of IgG, BSA and IgG/BSA mixtures at various mass ratios were measured, and the Langmuir-Freundlich model was used to fit the adsorption experimental data. The results indicated that IgG could be preferentially adsorbed by MMI resins, and MMI-B-4FF-100 resin with the pore size of 43.2 nm and the ligand density of 101 mol/g gel showed the best adsorption selectivity of IgG over BSA. In addition, binding behaviors of IgG and BSA in column were studied with MMI-B-4FF-100 at various pH values, and the optimized separation conditions were obtained as loading at pH 7.0 and elution at pH 4.0. Furthermore, IgG was effectively separated from IgG/BSA protein mixtures with MMI-B-4FF-100. The purities of IgG monomer reached above 95%, and the recovery kept about 90%. The results indicated that MMI-B-4FF-100 with high ligand density and moderate pore size would be a promising HCIC resin for IgG purification.
Keywords: hydrophobic charge-induction chromatography, competitive adsorption, immunoglobulin G, bovine serum albumin, adsorption isotherms, protein separation 3
1. Introduction Hydrophobic charge-induction chromatography (HCIC) was introduced in 1998 as a novel technique for selective capture of antibodies under physiological conditions [1, 2]. The basic characteristic of HCIC is the pH-dependent behavior of dual-mode ligands that combine hydrophobic and electrostatic interactions [3-6]. Target proteins can be adsorbed on uncharged ligands at neutral pH via hydrophobic interactions, and eluted via electrostatic repulsion between target protein and the charged ligand at acidic condition [7-10]. HCIC has been successfully applied to purify antibodies [5, 6, 9-11] and Fc-fusion proteins [12] with advantages of suitable selectivity, good reusability, mild elution condition and more flexible Clean-In-Place (CIP). However, compared with Protein A affinity chromatography, the separation selectivity of HCIC still needs to be improved. For instance, the presence of albumin in blood serum and cell culture media could affect the separation efficiency of immunoglobulin G (IgG) when using HCIC resins [13-15]. Better understanding on competitive adsorption between IgG and certain impurities in complicated feedstock is essential to illuminate adsorption mechanisms and improve separation selectivity with HCIC. The effects of impurities on target protein adsorption have been studied with various techniques and resins [16-24]. Finette et al [16] investigated the binding behavior of α1- proteinase inhibitor (α1-PI) in the presence of human serum albumin (HSA) in an anion-exchange packed bed. The results indicated that α1-PI had a higher 4
affinity for anion exchanger, and competitive adsorption was observed with the increase of HSA loading concentration. Xu et al [17] measured competitive adsorption of lysozyme and cytochrome c on different cation-exchangers, and they found that lysozyme significantly reduced cytochrome c adsorption. Morrison et al [18] investigated a selective desorption process to separate monomeric antibody (mAb) from associated aggregates and other impurities on ceramic hydroxyapatite. The separation performance was improved with 100% recovery of pure mAb after process optimization. In addition, confocal laser scanning microscopy (CLSM) has been used as a powerful tool to directly observe competitive adsorption behaviors between proteins within resin particles [19, 25, 26]. Competitive adsorption behaviors between IgG and bovine serum albumin (BSA) have also been investigated using some commercial HCIC resins. Tong et al [14, 27] studied IgG purification from serum albumin containing feedstock with MEP HyperCel resin, and found that the control of loading pH or additives might be an effective way to improve antibody purification with HCIC resins. Yan et al [28] investigated co-adsorption of IgG and BSA on Nuvia cPrime resins at various pH values and mass ratios. It was found that the adsorption capacity of both proteins decreased with the addition of another component and the resin preferred to adsorb IgG. Zhang et al [29] studied competitive adsorption of IgG and BSA with MEP HyperCel, and the results showed that although BSA adsorbed faster than IgG, part of the adsorbed BSA was gradually displaced by IgG as a result of competitive adsorption. In general, further investigations with series of HCIC 5
resins are needed in order to improve the understanding on competitive adsorption mechanism and enhance IgG selectivity. The objective of this work is to evaluate competitive adsorption behaviors of IgG/BSA mixtures on series of HCIC resins prepared in our lab. In our previous work [30], HCIC resins with different ligand densities and pore sizes using 2-mercapto-1-methyl-imidazole (MMI) as the ligand and agarose beads as the matrices were obtained, and the adsorption isotherms and kinetics of IgG on these resins were investigated. It was found that increase of ligand density and pore size could result in the improvement of adsorption affinity, capacity and effective pore diffusion. However, adsorption selectivity and separation efficiency of IgG from protein mixtures has not yet been studied. Therefore, in the present work the adsorption isotherms of IgG/BSA protein mixtures were investigated with series of MMI resins to explore competitive adsorption of binary protein components. The experimental
data
of
adsorption
equilibrium
was
evaluated
by
the
Langmuir-Freundlich model. In addition, binding and elution behaviors of the two proteins in column were studied at various pH values to optimize the IgG purification processes.
2. Materials and methods 2.1 Materials Three crossed-linked agarose gels with different agarose concentrations 6
(Bestarose 3.5HF, Bestarose 4FF and Bestarose 6FF) were purchased from Bestchrom Bio-Technology Co., Ltd. (Shanghai, China). 2-mercapto-1-methylimidazole (MMI) was purchased from Aladdin (Shanghai, China). Bovine γ-globulin (bovine IgG, Mw=153.1 kDa), which is bovine normal immunoglobulin G (IgG monomer > 95%) and bovine serum albumin (BSA, Mw=66.7 kDa, BSA monomer > 98%) were obtained from Sigma (Milwaukee, WI, USA). Other reagents were of analytical reagent grade and purchased locally. MMI resins with different ligand densities and pore sizes were prepared as published previously [30]. Bestarose 3.5HF, Bestarose 4FF, and Bestarose 6FF were used as the matrices with varying pore sizes. The pore size of agarose gels were measured by inverse size-exclusion chromatography and the average pore diameters of Bestarose-3.5HF, Bestarose-4FF and Bestarose-6FF are 47.9, 43.2 and 25.5 nm, respectively. MMI resins with different ligand densities were obtained by controlling activation and ligand coupling processes. Here, MMI-B-3.5HF-50 with ligand density of 50±1.7 mol/g gel, MMI-B-4FF-100 with ligand density of 101±2.3 mol/g gel and MMI-B-6FF-110 with ligand density of 111±2.5 mol/g gel were used.
2.2 Static adsorption experiments Adsorption isotherms of IgG, BSA and binary IgG/BSA protein mixtures with three MMI resins (MMI-B-3.5HF-50, MMI-B-4FF-100 and MMI-B-6FF-110) were measured with the procedure as published [30]. The protein mixtures with defined 7
total protein concentration of 40 mg/mL and different IgG/BSA mass ratios (1:1, 1:2, 1:3 and 1:4) was prepared and then diluted with 20 mM phosphate buffer (pH 7.0) to prepare protein mixture solutions with different concentrations. MMI resins were pre-equilibrated with 20 mM phosphate buffer (pH 7.0). 0.04 g drained resins were then added into 0.8 mL aliquots of IgG solution, BSA solution or IgG/BSA protein mixture which contained different concentrations of total protein (2-40 mg/mL) in 2 mL centrifuge tube. The solutions were agitated in a thermomixer (1000 rpm) at 25 °C for 5h. The samples were then filtered with 0.22 μm membrane filtration and the concentrations of IgG and BSA were analyzed by SEC-HPLC. The adsorption capacity for individual protein was calculated by mass balance. The Langmuir-Freundlich (LF) adsorption models [31, 32] were used to describe single-component adsorption equilibrium on MMI resins as,
q
qm ( Kc) n 1 ( Kc) n
(1)
where q and c are the equilibrium concentrations of protein in the stationary phase (mg/g gel) and in the liquid (mg/mL), respectively; qm is the saturated adsorption capacity (mg/g gel) and K is the association constant (mL/mg); n is the index of heterogeneity parameter. The extended Langmuir-Freundlich (ELF) model was applied to describe the binary adsorption equilibrium on MMI resins as,
q1
qm1 ( K1c1 )n1 1 ( K1c1 ) n1 ( K 2c2 ) n2
(2a) 8
q2
qm 2 ( K 2c2 )n2 1 ( K1c1 ) n1 ( K 2c2 ) n2
(2b)
where the subscript 1 and 2 represent IgG and BSA, respectively. qm1 and qm 2 are the saturation adsorption capacities, K1 and K2 are the average association constants, and n1 and n2 are the heterogeneity parameters for each component. Data and model fitting processes were performed as previously described [28].
2.3 Protein adsorption behaviors in the column The adsorption behaviors of IgG and BSA in the column were investigated with the ÄKTA explorer 100 system (GE Healthcare, Uppsala, Sweden). A 5-mm I.D. column (Tricorn 5/100, GE Healthcare, Uppsala, Sweden) was packed with 1.1 ml MMI-B-4FF-100 resin and equilibrated at the flow-rate of 0.5 ml/min with 10 CV equilibrium buffer (20 mM phosphate buffer, pH 7.0). IgG (1 mg/ml) or BSA (4 mg/ml) solutions at different pHs (pH 4.0, 5.0, 6.0, 7.0, 8.0, 8.5, 8.9) were loaded onto the column, and the unbound protein flowed through the column with the equilibrium buffer and the adsorbed protein was eluted by 0.1 M NaOH. The adsorption proportion of protein was calculated as follows,
PA (%) 100 -
flowthrough peak area 100 the sum of peak areas
(3)
2.4 IgG purification with MMI resins IgG separation from IgG/BSA protein mixtures using MMI-B-4FF-100 was 9
studied with the ÄKTA explorer 100 system and Tricorn 5/100 column. The protein mixture containing 1 mg/ml IgG and 4 mg/ml BSA was used as the loading feedstock to mimic the compositions in the serum. The flow rate was 0.5 ml/min. The column was equilibrated firstly with the equilibrium buffer (20 mM phosphate buffer, pH 7.0) and then protein mixtures were loaded. After sample loading, the column was washed by the equilibrium buffer. Finally, IgG was eluted with the acetate buffer (20 mM, pH 4.0). The chromatographic run was monitored on-line at 280 nm. The factions were collected and analyzed with the SDS-PAGE and SEC-HPLC. Based on SEC-HPLC, the IgG purity was calculated as the percentage of IgG monomer peak area in the total peak areas. The recovery of IgG was calculated according to the following equation,
R(%)
Elution amout ( IgG peak area volume) 100 Feed amount ( IgG peak area volume)
(4)
2.5 SDS-PAGE analysis The loading sample and collected fractions from IgG purification experiment were analyzed by 8% non-reducing SDS-PAGE gel. Protein solution was diluted to 0.5–2 mg/ml, and 5 μL protein solution was mixed with 20 μL sample treatment solution and heated for 3 min as the loading protein sample. Then 10 μL protein sample was loaded and the protein migration was performed under 220 V for 35 min. The gel was stained with Coomassie Blue R-250 and destained. The stained protein gel was imaged with the Gel Doc 2000 imaging system (Bio-Rad, Hercules, CA,
10
USA).
2.6 Determination of protein concentration by SEC -HPLC The concentrations of IgG and BSA in protein mixtures were measured by SEC -HPLC. The analysis was performed on LC3000 HPLC systems (Beijing Chuangxintongheng Science & Technology Co., Ltd., Beijing, China) using a TSK G3000SWXL column (7.8 mm × 30.0 cm, TOSOH, Japan). The mobile phase was 0.1 M sodium phosphate buffer (pH 6.7, containing 0.1 M Na2SO4) filtered by 0.22 m membrane filter and degassed. The flow rate of the mobile phase was 0.5 ml/min, and the sample injection volume was 20μL. The chromatographic run was monitored on-line at 280 nm by an UV detector. The retention time of IgG monomer was 16.5 min and that of BSA monomer was 18.5 min. Two peaks could be separated completely at the concentration range of 0-40 mg/ml. The standard curves of IgG and BSA were obtained with the individual protein solutions, and then used for the quantitative analysis of IgG and BSA concentrations in the IgG/BSA protein mixtures.
3. Results and discussion 3.1 Static competitive adsorption of IgG/BSA protein mixtures Based on our previous studies of IgG adsorption [30], three MMI resins (MMI-B-3.5HF-50, MMI-B-4FF-100 and MMI-B-6FF-110) showed favorable adsorption of IgG, and these resins were chosen to explore competitive adsorption of 11
binary IgG/BSA protein mixtures in the present work. The single-component adsorption isotherms (IgG and BSA) and binary coadsorption isotherms at various IgG/BSA initial mass ratios were measured, as shown in Fig. 1 to Fig. 3, respectively. Fig. 1 Fig. 2 Fig. 3 Competitive adsorption between IgG and BSA on MMI resins could be found at various IgG/BSA mass ratios. In general, the adsorption capacities of both proteins decreased with the addition of another component, which indicates that IgG and BSA might be adsorbed at the same binding sites on MMI resins. For low protein concentrations (<2 mg/mL), the adsorption capacities of both proteins under various IgG/BSA mass ratios were comparable to that of the single-component profile. This might be because the ligands were not fully occupied by these protein molecules. However, the adsorption differentiations for IgG/BSA protein mixtures became obvious under high protein concentrations. With MMI-B-4FF-100 as an example, IgG adsorption was influenced obviously with the presence of BSA. The saturated adsorption capacity of IgG was around 70 mg/g gel for the single-component adsorption, which dropped to 50 mg/g gel when equivalent BSA was added, and it further dropped to 30 mg/g gel when 4-fold BSA was added. Correspondingly, the adsorption capacities of BSA also declined in the binary adsorption which showed more significant changes than IgG. The saturated adsorption capacity of BSA was 12
around 30 mg/g gel in the absence of IgG, but dropped to 16 mg/g gel when the IgG/BSA ratio was 1:3, and it further dropped to 13 mg/g gel with IgG/BSA mass ratio increased to 1:1. In addition, it was found that the total adsorption capacity of IgG and BSA in binary adsorption was much higher than that of single BSA component, and it increased as the IgG/BSA mass ratio changed from 1:4 to 1:1, which suggests that some BSA was displaced by IgG. The results demonstrate the existence of competitive adsorption between IgG and BSA, and a preferential adsorption and selectivity of IgG to BSA was found under the conditions tested. The single-component adsorption isotherms of IgG and BSA were correlated with Langmuir-Freundlich model (eq. (1)), and the adsorption parameters were determined and listed in Table 1. The Extended Langmuir-Freundlich model (eq. (2)) was used to predict the isotherms of binary adsorption at various IgG/BSA initial mass ratios (1:1, 1:2, 1:3 and 1:4) using the parameters obtained from the single-component system. Specific calculation processes could be found in reference [28]. The Langmuir-Freundlich model is a combination of Langmuir and Freundlich isotherms and shows a versatile expression which can be used to study both Langmuir-type and Freundlich-type adsorption behaviors. The importance is the introduction of heterogeneity index (n) which can well represent the type and extent of cooperative binding interactions [28, 30]. As shown in Figs. 1 to 3, the models could correlate the single-component adsorption isotherms and predict the binary adsorption isotherms. As shown in Table 1, the n values of both proteins were around 13
0.83-0.87, indicating that the adsorption heterogeneity was similar. The qm and K values were shown in two bases, protein weight (mg) and number (mol). It could be found that based on protein weight, the qm values of IgG were obviously higher than those of BSA. However, based on protein number, the qm values of IgG and BSA were quite similar. The results indicated at the saturated adsorption situation, MMI resins could adsorb nearly same amount of IgG as BSA. But the equilibrium adsorption capacities of IgG were obviously higher than those of BSA at the same protein concentrations in the liquid. Take MMI-B-4FF-100 for instance, when the equilibrium protein concentrations in the liquid was about 2 mg/mL, the equilibrium adsorption capacities of IgG and BSA were around 39.11 mg/g gel and 6.81 mg/g gel, respectively. Even based on protein number, the equilibrium adsorption capacity of IgG was 2.5-time higher than that of BSA. In addition, the K values of IgG were much higher than those of BSA, especially based on the protein number, indicating that the affinity between MMI resin and IgG were much stronger than that of BSA.
Table1. Parameters of adsorption isotherms correlated by Langmuir-Freundlich model MMI resins
MMI-B-3.5HF-50
MMI-B-4FF-100
MMI-B-6FF-110
qm1a (mg/g gel)
63.75±3.67
93.51±5.23
86.63±5.01
K1b (mL/mg)
0.325±0.011
0.355±0.015
0.515±0.021
qm1a (μmol/g gel)
0.4163±0.0239
0.6107±0.0342
0.5658±0.0327
K1b (mL/μmol)
49.75±1.68
54.35±2.30
78.85±3.21
n1c
0.87±0.02
0.83±0.03
0.87±0.03
RMSE1d
0.128
0.179
0.072 14
qm2 a (mg/g gel)
43.12±5.33
43.69±4.12
45.33±4.23
K2 b (mL/mg)
0.078±0.008
0.144±0.009
0.205±0.012
qm2 a (μmol/g gel)
0.6465±0.0012
0.6550±0.0022
0.6796±0.0634
K2 b (mL/μmol)
5.20±0.53
9.60±0.60
13.67±0.80
n2 c
0.86±0.03
0.86±0.02
0.84±0.02
0.222
0.306
0.133
RMSE2
d
a
qm1 and qm2: saturation adsorption capacities of IgG and BSA, respectively;
b
K1 and K2: average association constants of IgG and BSA, respectively;
c
n1 and n2: heterogeneity parameters of IgG and BSA, respectively.
d
RMSE1 and RMSE2: root mean square error of adsorption isotherms of IgG and BSA,
respectively. The possible reasons for competitive adsorption between IgG and BSA on MMI resins can be attributed to the difference of partial hydrophobicity on the protein surface and molecular size. As shown with molecular simulation [7, 8, 33], there are some hydrophobic pockets on the surface of IgG, which might be more suitable for the binding to hydrophobic ligands such as MMI. Therefore, a stronger attraction might exist between IgG and MMI ligands due to the complicated hydrophobic interactions when comparing to BSA. Meanwhile, BSA with smaller molecular size, higher concentration and faster mobility can transfer and be adsorbed onto binding sites faster than IgG. However, IgG molecules would compete for the limited binding sites due to stronger protein-ligand attraction between IgG and HCIC ligands [29], which leads to the competitive adsorption behaviors.
15
3.2 Comparison of different resins As listed in Table 1, the qm value of IgG with MMI-B-4FF-100 resin was the highest among three resins tested, while that of BSA were similar, which suggests that MMI-B-4FF-100 exhibits the best binding ability of IgG and adsorption selectivity of IgG to BSA. To further evaluate the co-adsorption behaviors of IgG and BSA in binary systems, the molar ratios of IgG/BSA on MMI resins and those in the liquid phase were compared, and the results are shown in Fig. 4. It could be found that the molar ratios of IgG/BSA in the stationary phase were more than 5 times higher than those in the liquid phase, which indicates that IgG was more favorably bound to the MMI resins than BSA. For MMI-B-4FF-100, even 10 times of IgG/BSA molar ratios in the stationary phase than those in the liquid phase could be obtained. The results indicate that among the three MMI resins tested, MMI-B-4FF-100 possessed the most excellent adsorption ability as well as preferential adsorption of IgG to BSA. In addition, a linear relation (y=k*x) could be found between the molar ratios of IgG/BSA in the stationary phase and those in liquid phase, which could be used to describe and compare the preferential adsorption behavior of IgG to BSA. The correlated values of k and R2 are listed in Table 2. The k value of MMI-B-4FF-100 was the highest, which reveals the outstanding adsorption selectively of IgG to BSA. Fig. 4
Table 2. Parameters of linear fitting between the molar ratio of IgG/BSA in the stationary phase and in liquid phase with different MMI resins 16
a
MMI resins
MMI-B-3.5HF-50
MMI-B-4FF-100
MMI-B-6FF-110
ka
5.38
8.18
4.74
R2 b
0.9774
0.9403
0.9693
k means the slope of a linear relation between the molar ratios of IgG/BSA in the
stationary phase and those in liquid phase; b
R2 means the fitting degree of linear relation.
The results indicate that MMI-B-4FF-100 seems to be the most satisfactory resin to separate IgG selectively. The possible reasons are described as follows. As reported in our previous work [30], high ligand density on the internal surface of the MMI resin is necessary to provide enough attractive interactions to capture the protein. The pore surface ligand density (PSLD, ligand number/nm2) was proposed in our previous work [34], which is based on the hypothesis that the internal pore of resin can be regarded as idealized cylinders and the ligand distribute evenly on the internal pore [35]. The PSLD values of the three MMI resins tested were calculated as 0.77, 1.36 and 0.89 ligand number/nm2, respectively. It could be found that the PSLD value of the MMI-B-4FF-100 resin was the largest, which could provide higher accessibility and stronger hydrophobic interactions for IgG binding. Meanwhile, BSA was smaller and less hydrophobic than IgG, the attraction existed between BSA and MMI resin was weaker so the influence of PSLD on mass transfer and adsorption of BSA with MMI resin was less important. In general, among three MMI resins tested in the present work, MMI-B-4FF-100 showed the best adsorption selectivity of IgG to BSA 17
in the IgG/BSA binary adsorption, thus this resin would be the most suitable resin for the purification of IgG from the BSA-containing feedstock.
3.3 Protein adsorption behaviors in the column Protein adsorption on HCIC resins is typically pH-dependent [27, 36], and the control of pH is an effective way to adjust the adsorption of IgG and BSA on the MMI resins. Therefore, the adsorption behaviors of IgG and BSA in the column with MMI-B-4FF-100 resin were determined further at varying pHs to explore the optimal conditions for IgG separation from BSA-containing feedstock. The chromatographic results are shown in Fig. 5, and the adsorption proportions and the relative selectivity at different pH conditions are compared in Fig. 6. Fig. 5 Fig. 6 As shown in Fig. 6, remarkable difference could be found for IgG and BSA adsorption in the column with MMI-B-4FF-100 at varying pHs. For BSA, the adsorption amount increased sharply from 10.02% to 41.11 % as the pH increased from 4 to 5, and then decreased slightly at the pH range of 5~6, which further decreased to 3.72% with the increase of pH to 7. Compared with BSA, IgG kept high adsorption proportion at a relative wide range of pH 6~8, while adsorption reduced gradually as the pH changed from 6 to 4 or from 8 to 8.9. It could be found that IgG was adsorbed efficiently with the adsorption proportion of ~100% at pH 7, while BSA 18
was partially adsorbed with a maximum adsorption proportion of about 40% at pH 5.0. The pIs of IgG and BSA were around 6.8~10 and 4.8, respectively, and both IgG and BSA reached the maximum adsorption at the pHs around their individual pI. This phenomenon was consistent with that reported by Tong et al [27] and Yan et al [36]. Generally, protein molecule has relative small hydrodynamic diameter around the pI due to few net charges and thin hydrated layer on the surface, which would be favorable for the protein adsorption on hydrophobic interaction-based resins, such as MMI resins used in the present work [30]. The results of protein adsorption behaviors in the column indicate that pH control is a good way to improve IgG separation efficiency from binary IgG/BSA protein mixtures. At pH 7.0, almost all IgG was adsorbed on MMI-B-4FF-100 and most BSA was washed out, which indicates that IgG could be adsorbed selectively from the IgG/BSA protein mixtures. Meanwhile, near 100% IgG was found in non-adsorbed flowthrough fraction at pH 4.0 due to strong electrostatic repulsion between the positively-charged proteins and MMI ligands at acid condition. Therefore, the separation process of IgG from BSA-containing feedstock with MMI resin could be set as the adsorption-elution mode: equilibrium and loading at pH 7.0 to ensure the adsorption of IgG and the flowthrough of BSA, and then elution at pH 4.0 to obtain high recovery of IgG.
3.4 IgG separation from IgG/BSA protein mixture with MMI resins 19
The MMI-B-4FF-100 resin was used to separate IgG from BSA-containing feedstock under optimized separation conditions (loading at pH 7.0 and elution at pH 4.0). A mixture of IgG (1 mg/mL) and BSA (4 mg/mL) was used as a model feedstock to mimic the composition in serum. The chromatograms at various loading volumes (1, 2, 5, 10 mL) are shown in Fig. 7. The flowthrough and elution factions were collected and analyzed by SDS-PAGE and SEC-HPLC. The results of SDS-PAGE are also shown in Fig. 7. It can be found that the main compound in flowthrough fraction was BSA with few IgG detected, which indicates that IgG could be well adsorbed with MMI-B-4FF-100 resin at pH 7.0. Meanwhile, almost no BSA was detected in the elution fraction, which demonstrates that IgG could be separated efficiently from the binary IgG/BSA protein mixtures under the present separation condition. Fig. 7 The purities and recoveries of IgG at various loading volumes were determined by SEC-HPLC. For all the separation processes at various loading volumes, no BSA but only IgG monomer and few IgG aggregate were detected in the elution fractions. The purities of IgG monomer were above 95%, and the recovery of IgG monomer kept around 85-90% at various loading volumes. The results demonstrate that the MMI-B-4FF-100 resin can efficiently separate IgG from binary IgG/BSA protein mixtures. Compared to similar separation process with the commercial HCIC resin MEP-HyperCel reported by Tong et al [14, 27] (the purity of IgG was 62.9%), the 20
present process with the MMI resin was more efficient. For MEP-HyperCel resin, some additives like NaCl [27] or caprylate sodium [14] should be added in the loading buffer or washing buffer to improve IgG purity to reach about ~95%. Therefore, the MMI-B-4FF-100 resin with high ligand density and moderate pore size would be a promising HCIC resin for IgG purification. The results indicate that the combination of three factors, ligand density, pore size and hydrophobicity of ligand, determine together the separation performance of HCIC resins.
4. Conclusions In the present work, competitive adsorption behaviors between IgG and BSA were investigated with three MMI resins to study adsorption mechanism and enhance separation efficiency of IgG from the BSA-containing feedstock. The adsorption isotherms of single component and binary IgG/BSA protein mixtures at various mass ratios were measured, and the Langmuir-Freundlich adsorption model was used to fit the experimental data. The results showed that IgG could be adsorbed preferentially by MMI resins, and MMI-B-4FF-100 with moderate pore size and high ligand density exhibited the best adsorption selectivity of IgG over BSA. In addition, the binding behaviors of IgG and BSA in the column with MMI-B-4FF-100 were studied at various pHs to determine the optimal IgG separation conditions. Finally, IgG was separated from IgG/BSA protein mixtures using MMI-B-4FF-100, and high purity and recovery of IgG were obtained. The results indicated that suitable pore size and 21
ligand density of resins, and the control of pH are three critical factors to adjust the binding selectivity of IgG and improve separation efficiency of HCIC processes. MMI-B-4FF-100 with high ligand density and moderate pore size would be a promising HCIC resin for IgG purification.
Acknowledgments This work was supported by the National Natural Science Foundation of China and the China Postdoctoral Science Foundation (512100-X91603).
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[24] Y. Tao, N. Chen, G. Carta, G. Ferreira, D. Robbins, Modeling multicomponent adsorption of monoclonal antibody charge variants in cation exchange columns, AIChE Journal, 58 (2011) 2503-2511. [25] T. Linden, A. Ljungloef, M.R. Kula, J. Thoemmes, Visualizing two‐component protein diffusion in porous adsorbents by confocal scanning laser microscopy, Biotechnology and bioengineering, 65 (1999) 622-630. [26] M.M.H. El-Sayed, H.A. Chase, Confocal microscopy study of uptake kinetics of α-lactalbumin and β-lactoglobulin onto the cation-exchanger SP Sepharose FF, Journal of Separation Science, 32 (2009) 3246–3256. [27] H.F. Tong, D.Q. Lin, X.M. Yuan, S.J. Yao, Enhancing IgG purification from serum albumin containing feedstock with hydrophobic charge-induction chromatography, Journal of Chromatography A, (2012). [28] J. Yan, Q.L. Zhang, D.Q. Lin, S.J. Yao, Coadsorption of Human Immunoglobulin G and Bovine Serum Albumin on a p-Aminohippuric Acid Based Mixed-Mode Resin, Journal of Chemical & Engineering Data, 61 (2015) 151-159. [29] Q. Zhang, F. Schimpf, H.L. Lu, D.Q. Lin, S.J. Yao, Binary Adsorption Processes of Albumin and Immunoglobulin on Hydrophobic Charge-Induction Resins, Journal of Chemical & Engineering Data, (2016). [30] H.L. Lu, D.-Q. Lin, D. Gao, S.-J. Yao, Evaluation of immunoglobulin adsorption on the hydrophobic charge-induction resins with different ligand densities and 26
pore sizes, Journal of Chromatography A, 1278 (2013) 61-68. [31] T. Cano, N. Offringa, R.C. Willson, The effectiveness of three multi-component binding models in describing the binary competitive equilibrium adsorption of two cytochrome b5 mutants, Journal of Chromatography A, 1144 (2007) 197-202. [32] T. Cano, N.D. Offringa, R.C. Willson, Competitive ion-exchange adsorption of proteins: Competitive isotherms with controlled competitor concentration, Journal of Chromatography A, 1079 (2005) 116-126. [33] H.Y. Wang, D.Q. Lin, S.J. Yao, Molecular Simulation of the Interactions between 4-Mercaptoethyl-pyridine Ligand and IgG, Acta Chim Sinica, 68 (2010) 1597-1602. [34] C.X. Wang, D.Q. Lin, T. Liu, S.J. Yao, Hydrophobic charge-induction chromatographic resin with 5-aminobenzimidazol ligand: effects of ligand density on protein adsorption, Separation Science & Technology, (2016). [35] B.D. Bowes, H. Koku, K.J. Czymmek, A.M. Lenhoff, Protein adsorption and transport in dextran-modified ion-exchange media. I: Adsorption, Journal of Chromatography A, 1216 (2009) 7774-7784. [36] J. Yan, Q.L. Zhang, H.F. Tong, D.Q. Lin, S.J. Yao, Hydrophobic charge-induction resin with 5-aminobenzimidazol as the functional ligand: preparation, protein adsorption and immunoglobulin G purification, Journal of Separation Science, 38 (2015) 2387-2393. 27
28
Figure Captions Fig. 1 Adsorption isotherms of single and protein mixtures on MMI-B-3.5HF-50. (a) IgG; (b) BSA Fig. 2 Adsorption isotherms of single and protein mixtures on MMI-B-4FF-100. (a) IgG; (b) BSA Fig. 3 Adsorption isotherms of single and protein mixtures on MMI-B-6FF-110. (a) IgG; (b) BSA Fig. 4 Comparison of molar ratios of IgG and BSA on the resins and in liquid phase Fig. 5 Chromatograms of IgG (a) and BSA (b) in the column with MMI-B-4FF-100 resin at various pHs. For all curves, the first peak at about 5~10 min was the flowthrough peak, and the second peak at about 25 min was the CIP peak. Fig. 6 IgG and BSA adsorption in the column with MMI-B-4FF-100 resin as the function of loading pH Fig. 7 Chromatograms of IgG separation from IgG/BSA mixtures in the column with MMI-B-4FF-100. Insert: SDS-PAGE analysis of the fractions. FT and EL mean the fractions from flowthrough and elution pools, respectively.
29
S1369-703X(16)30338-2 http://dx.doi.org/doi:10.1016/j.bej.2016.12.005 BEJ 6612
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
5-9-2016 1-12-2016 3-12-2016
Please cite this article as: Hui-Li Lu, Dong-Qiang Lin, Qi-Lei Zhang, Shan-Jing Yao, Evaluation on adsorption selectivity of immunoglobulin G with 2-mercapto-1-methylimidazole-based hydrophobic charge-induction resins, Biochemical Engineering Journal http://dx.doi.org/10.1016/j.bej.2016.12.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Evaluation on adsorption selectivity of immunoglobulin G with 2-mercapto-1-methyl-imidazole-based hydrophobic charge-induction resins
Hui-Li Lu, Dong-Qiang Lin*, Qi-Lei Zhang, Shan-Jing Yao*
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
Corresponding author: Prof. Dong-Qiang Lin and Prof. Shan-Jing Yao College of Chemical and Biological Engineering Zhejiang University Hangzhou 310027 China Fax: +86-571-87951982 E-mail: [email protected]; [email protected]
1
Research highlights ► 2-mercapto-1-methyl-imidazole-based hydrophobic charge-induction resins were evaluated. ► Competitive adsorption behaviors between immunoglobulin G and bovine serum albumin were investigated ► Resins with moderate pore size and high ligand density exhibited the best adsorption selectivity. ► Immunoglobulin G was preferentially adsorbed. ► Immunoglobulin G was separated from protein mixtures effectively.
2
ABSTRACT
Hydrophobic
charge-induction
chromatography
(HCIC)
with
2-mercapto-1-methyl-imidazole (MMI) ligands shows promising results in antibody purification. In this study, competitive adsorption processes between bovine immunoglobulin G (IgG) and bovine serum albumin (BSA) were investigated with MMI resins. Adsorption isotherms of IgG, BSA and IgG/BSA mixtures at various mass ratios were measured, and the Langmuir-Freundlich model was used to fit the adsorption experimental data. The results indicated that IgG could be preferentially adsorbed by MMI resins, and MMI-B-4FF-100 resin with the pore size of 43.2 nm and the ligand density of 101 mol/g gel showed the best adsorption selectivity of IgG over BSA. In addition, binding behaviors of IgG and BSA in column were studied with MMI-B-4FF-100 at various pH values, and the optimized separation conditions were obtained as loading at pH 7.0 and elution at pH 4.0. Furthermore, IgG was effectively separated from IgG/BSA protein mixtures with MMI-B-4FF-100. The purities of IgG monomer reached above 95%, and the recovery kept about 90%. The results indicated that MMI-B-4FF-100 with high ligand density and moderate pore size would be a promising HCIC resin for IgG purification.
Keywords: hydrophobic charge-induction chromatography, competitive adsorption, immunoglobulin G, bovine serum albumin, adsorption isotherms, protein separation 3
1. Introduction Hydrophobic charge-induction chromatography (HCIC) was introduced in 1998 as a novel technique for selective capture of antibodies under physiological conditions [1, 2]. The basic characteristic of HCIC is the pH-dependent behavior of dual-mode ligands that combine hydrophobic and electrostatic interactions [3-6]. Target proteins can be adsorbed on uncharged ligands at neutral pH via hydrophobic interactions, and eluted via electrostatic repulsion between target protein and the charged ligand at acidic condition [7-10]. HCIC has been successfully applied to purify antibodies [5, 6, 9-11] and Fc-fusion proteins [12] with advantages of suitable selectivity, good reusability, mild elution condition and more flexible Clean-In-Place (CIP). However, compared with Protein A affinity chromatography, the separation selectivity of HCIC still needs to be improved. For instance, the presence of albumin in blood serum and cell culture media could affect the separation efficiency of immunoglobulin G (IgG) when using HCIC resins [13-15]. Better understanding on competitive adsorption between IgG and certain impurities in complicated feedstock is essential to illuminate adsorption mechanisms and improve separation selectivity with HCIC. The effects of impurities on target protein adsorption have been studied with various techniques and resins [16-24]. Finette et al [16] investigated the binding behavior of α1- proteinase inhibitor (α1-PI) in the presence of human serum albumin (HSA) in an anion-exchange packed bed. The results indicated that α1-PI had a higher 4
affinity for anion exchanger, and competitive adsorption was observed with the increase of HSA loading concentration. Xu et al [17] measured competitive adsorption of lysozyme and cytochrome c on different cation-exchangers, and they found that lysozyme significantly reduced cytochrome c adsorption. Morrison et al [18] investigated a selective desorption process to separate monomeric antibody (mAb) from associated aggregates and other impurities on ceramic hydroxyapatite. The separation performance was improved with 100% recovery of pure mAb after process optimization. In addition, confocal laser scanning microscopy (CLSM) has been used as a powerful tool to directly observe competitive adsorption behaviors between proteins within resin particles [19, 25, 26]. Competitive adsorption behaviors between IgG and bovine serum albumin (BSA) have also been investigated using some commercial HCIC resins. Tong et al [14, 27] studied IgG purification from serum albumin containing feedstock with MEP HyperCel resin, and found that the control of loading pH or additives might be an effective way to improve antibody purification with HCIC resins. Yan et al [28] investigated co-adsorption of IgG and BSA on Nuvia cPrime resins at various pH values and mass ratios. It was found that the adsorption capacity of both proteins decreased with the addition of another component and the resin preferred to adsorb IgG. Zhang et al [29] studied competitive adsorption of IgG and BSA with MEP HyperCel, and the results showed that although BSA adsorbed faster than IgG, part of the adsorbed BSA was gradually displaced by IgG as a result of competitive adsorption. In general, further investigations with series of HCIC 5
resins are needed in order to improve the understanding on competitive adsorption mechanism and enhance IgG selectivity. The objective of this work is to evaluate competitive adsorption behaviors of IgG/BSA mixtures on series of HCIC resins prepared in our lab. In our previous work [30], HCIC resins with different ligand densities and pore sizes using 2-mercapto-1-methyl-imidazole (MMI) as the ligand and agarose beads as the matrices were obtained, and the adsorption isotherms and kinetics of IgG on these resins were investigated. It was found that increase of ligand density and pore size could result in the improvement of adsorption affinity, capacity and effective pore diffusion. However, adsorption selectivity and separation efficiency of IgG from protein mixtures has not yet been studied. Therefore, in the present work the adsorption isotherms of IgG/BSA protein mixtures were investigated with series of MMI resins to explore competitive adsorption of binary protein components. The experimental
data
of
adsorption
equilibrium
was
evaluated
by
the
Langmuir-Freundlich model. In addition, binding and elution behaviors of the two proteins in column were studied at various pH values to optimize the IgG purification processes.
2. Materials and methods 2.1 Materials Three crossed-linked agarose gels with different agarose concentrations 6
(Bestarose 3.5HF, Bestarose 4FF and Bestarose 6FF) were purchased from Bestchrom Bio-Technology Co., Ltd. (Shanghai, China). 2-mercapto-1-methylimidazole (MMI) was purchased from Aladdin (Shanghai, China). Bovine γ-globulin (bovine IgG, Mw=153.1 kDa), which is bovine normal immunoglobulin G (IgG monomer > 95%) and bovine serum albumin (BSA, Mw=66.7 kDa, BSA monomer > 98%) were obtained from Sigma (Milwaukee, WI, USA). Other reagents were of analytical reagent grade and purchased locally. MMI resins with different ligand densities and pore sizes were prepared as published previously [30]. Bestarose 3.5HF, Bestarose 4FF, and Bestarose 6FF were used as the matrices with varying pore sizes. The pore size of agarose gels were measured by inverse size-exclusion chromatography and the average pore diameters of Bestarose-3.5HF, Bestarose-4FF and Bestarose-6FF are 47.9, 43.2 and 25.5 nm, respectively. MMI resins with different ligand densities were obtained by controlling activation and ligand coupling processes. Here, MMI-B-3.5HF-50 with ligand density of 50±1.7 mol/g gel, MMI-B-4FF-100 with ligand density of 101±2.3 mol/g gel and MMI-B-6FF-110 with ligand density of 111±2.5 mol/g gel were used.
2.2 Static adsorption experiments Adsorption isotherms of IgG, BSA and binary IgG/BSA protein mixtures with three MMI resins (MMI-B-3.5HF-50, MMI-B-4FF-100 and MMI-B-6FF-110) were measured with the procedure as published [30]. The protein mixtures with defined 7
total protein concentration of 40 mg/mL and different IgG/BSA mass ratios (1:1, 1:2, 1:3 and 1:4) was prepared and then diluted with 20 mM phosphate buffer (pH 7.0) to prepare protein mixture solutions with different concentrations. MMI resins were pre-equilibrated with 20 mM phosphate buffer (pH 7.0). 0.04 g drained resins were then added into 0.8 mL aliquots of IgG solution, BSA solution or IgG/BSA protein mixture which contained different concentrations of total protein (2-40 mg/mL) in 2 mL centrifuge tube. The solutions were agitated in a thermomixer (1000 rpm) at 25 °C for 5h. The samples were then filtered with 0.22 μm membrane filtration and the concentrations of IgG and BSA were analyzed by SEC-HPLC. The adsorption capacity for individual protein was calculated by mass balance. The Langmuir-Freundlich (LF) adsorption models [31, 32] were used to describe single-component adsorption equilibrium on MMI resins as,
q
qm ( Kc) n 1 ( Kc) n
(1)
where q and c are the equilibrium concentrations of protein in the stationary phase (mg/g gel) and in the liquid (mg/mL), respectively; qm is the saturated adsorption capacity (mg/g gel) and K is the association constant (mL/mg); n is the index of heterogeneity parameter. The extended Langmuir-Freundlich (ELF) model was applied to describe the binary adsorption equilibrium on MMI resins as,
q1
qm1 ( K1c1 )n1 1 ( K1c1 ) n1 ( K 2c2 ) n2
(2a) 8
q2
qm 2 ( K 2c2 )n2 1 ( K1c1 ) n1 ( K 2c2 ) n2
(2b)
where the subscript 1 and 2 represent IgG and BSA, respectively. qm1 and qm 2 are the saturation adsorption capacities, K1 and K2 are the average association constants, and n1 and n2 are the heterogeneity parameters for each component. Data and model fitting processes were performed as previously described [28].
2.3 Protein adsorption behaviors in the column The adsorption behaviors of IgG and BSA in the column were investigated with the ÄKTA explorer 100 system (GE Healthcare, Uppsala, Sweden). A 5-mm I.D. column (Tricorn 5/100, GE Healthcare, Uppsala, Sweden) was packed with 1.1 ml MMI-B-4FF-100 resin and equilibrated at the flow-rate of 0.5 ml/min with 10 CV equilibrium buffer (20 mM phosphate buffer, pH 7.0). IgG (1 mg/ml) or BSA (4 mg/ml) solutions at different pHs (pH 4.0, 5.0, 6.0, 7.0, 8.0, 8.5, 8.9) were loaded onto the column, and the unbound protein flowed through the column with the equilibrium buffer and the adsorbed protein was eluted by 0.1 M NaOH. The adsorption proportion of protein was calculated as follows,
PA (%) 100 -
flowthrough peak area 100 the sum of peak areas
(3)
2.4 IgG purification with MMI resins IgG separation from IgG/BSA protein mixtures using MMI-B-4FF-100 was 9
studied with the ÄKTA explorer 100 system and Tricorn 5/100 column. The protein mixture containing 1 mg/ml IgG and 4 mg/ml BSA was used as the loading feedstock to mimic the compositions in the serum. The flow rate was 0.5 ml/min. The column was equilibrated firstly with the equilibrium buffer (20 mM phosphate buffer, pH 7.0) and then protein mixtures were loaded. After sample loading, the column was washed by the equilibrium buffer. Finally, IgG was eluted with the acetate buffer (20 mM, pH 4.0). The chromatographic run was monitored on-line at 280 nm. The factions were collected and analyzed with the SDS-PAGE and SEC-HPLC. Based on SEC-HPLC, the IgG purity was calculated as the percentage of IgG monomer peak area in the total peak areas. The recovery of IgG was calculated according to the following equation,
R(%)
Elution amout ( IgG peak area volume) 100 Feed amount ( IgG peak area volume)
(4)
2.5 SDS-PAGE analysis The loading sample and collected fractions from IgG purification experiment were analyzed by 8% non-reducing SDS-PAGE gel. Protein solution was diluted to 0.5–2 mg/ml, and 5 μL protein solution was mixed with 20 μL sample treatment solution and heated for 3 min as the loading protein sample. Then 10 μL protein sample was loaded and the protein migration was performed under 220 V for 35 min. The gel was stained with Coomassie Blue R-250 and destained. The stained protein gel was imaged with the Gel Doc 2000 imaging system (Bio-Rad, Hercules, CA,
10
USA).
2.6 Determination of protein concentration by SEC -HPLC The concentrations of IgG and BSA in protein mixtures were measured by SEC -HPLC. The analysis was performed on LC3000 HPLC systems (Beijing Chuangxintongheng Science & Technology Co., Ltd., Beijing, China) using a TSK G3000SWXL column (7.8 mm × 30.0 cm, TOSOH, Japan). The mobile phase was 0.1 M sodium phosphate buffer (pH 6.7, containing 0.1 M Na2SO4) filtered by 0.22 m membrane filter and degassed. The flow rate of the mobile phase was 0.5 ml/min, and the sample injection volume was 20μL. The chromatographic run was monitored on-line at 280 nm by an UV detector. The retention time of IgG monomer was 16.5 min and that of BSA monomer was 18.5 min. Two peaks could be separated completely at the concentration range of 0-40 mg/ml. The standard curves of IgG and BSA were obtained with the individual protein solutions, and then used for the quantitative analysis of IgG and BSA concentrations in the IgG/BSA protein mixtures.
3. Results and discussion 3.1 Static competitive adsorption of IgG/BSA protein mixtures Based on our previous studies of IgG adsorption [30], three MMI resins (MMI-B-3.5HF-50, MMI-B-4FF-100 and MMI-B-6FF-110) showed favorable adsorption of IgG, and these resins were chosen to explore competitive adsorption of 11
binary IgG/BSA protein mixtures in the present work. The single-component adsorption isotherms (IgG and BSA) and binary coadsorption isotherms at various IgG/BSA initial mass ratios were measured, as shown in Fig. 1 to Fig. 3, respectively. Fig. 1 Fig. 2 Fig. 3 Competitive adsorption between IgG and BSA on MMI resins could be found at various IgG/BSA mass ratios. In general, the adsorption capacities of both proteins decreased with the addition of another component, which indicates that IgG and BSA might be adsorbed at the same binding sites on MMI resins. For low protein concentrations (<2 mg/mL), the adsorption capacities of both proteins under various IgG/BSA mass ratios were comparable to that of the single-component profile. This might be because the ligands were not fully occupied by these protein molecules. However, the adsorption differentiations for IgG/BSA protein mixtures became obvious under high protein concentrations. With MMI-B-4FF-100 as an example, IgG adsorption was influenced obviously with the presence of BSA. The saturated adsorption capacity of IgG was around 70 mg/g gel for the single-component adsorption, which dropped to 50 mg/g gel when equivalent BSA was added, and it further dropped to 30 mg/g gel when 4-fold BSA was added. Correspondingly, the adsorption capacities of BSA also declined in the binary adsorption which showed more significant changes than IgG. The saturated adsorption capacity of BSA was 12
around 30 mg/g gel in the absence of IgG, but dropped to 16 mg/g gel when the IgG/BSA ratio was 1:3, and it further dropped to 13 mg/g gel with IgG/BSA mass ratio increased to 1:1. In addition, it was found that the total adsorption capacity of IgG and BSA in binary adsorption was much higher than that of single BSA component, and it increased as the IgG/BSA mass ratio changed from 1:4 to 1:1, which suggests that some BSA was displaced by IgG. The results demonstrate the existence of competitive adsorption between IgG and BSA, and a preferential adsorption and selectivity of IgG to BSA was found under the conditions tested. The single-component adsorption isotherms of IgG and BSA were correlated with Langmuir-Freundlich model (eq. (1)), and the adsorption parameters were determined and listed in Table 1. The Extended Langmuir-Freundlich model (eq. (2)) was used to predict the isotherms of binary adsorption at various IgG/BSA initial mass ratios (1:1, 1:2, 1:3 and 1:4) using the parameters obtained from the single-component system. Specific calculation processes could be found in reference [28]. The Langmuir-Freundlich model is a combination of Langmuir and Freundlich isotherms and shows a versatile expression which can be used to study both Langmuir-type and Freundlich-type adsorption behaviors. The importance is the introduction of heterogeneity index (n) which can well represent the type and extent of cooperative binding interactions [28, 30]. As shown in Figs. 1 to 3, the models could correlate the single-component adsorption isotherms and predict the binary adsorption isotherms. As shown in Table 1, the n values of both proteins were around 13
0.83-0.87, indicating that the adsorption heterogeneity was similar. The qm and K values were shown in two bases, protein weight (mg) and number (mol). It could be found that based on protein weight, the qm values of IgG were obviously higher than those of BSA. However, based on protein number, the qm values of IgG and BSA were quite similar. The results indicated at the saturated adsorption situation, MMI resins could adsorb nearly same amount of IgG as BSA. But the equilibrium adsorption capacities of IgG were obviously higher than those of BSA at the same protein concentrations in the liquid. Take MMI-B-4FF-100 for instance, when the equilibrium protein concentrations in the liquid was about 2 mg/mL, the equilibrium adsorption capacities of IgG and BSA were around 39.11 mg/g gel and 6.81 mg/g gel, respectively. Even based on protein number, the equilibrium adsorption capacity of IgG was 2.5-time higher than that of BSA. In addition, the K values of IgG were much higher than those of BSA, especially based on the protein number, indicating that the affinity between MMI resin and IgG were much stronger than that of BSA.
Table1. Parameters of adsorption isotherms correlated by Langmuir-Freundlich model MMI resins
MMI-B-3.5HF-50
MMI-B-4FF-100
MMI-B-6FF-110
qm1a (mg/g gel)
63.75±3.67
93.51±5.23
86.63±5.01
K1b (mL/mg)
0.325±0.011
0.355±0.015
0.515±0.021
qm1a (μmol/g gel)
0.4163±0.0239
0.6107±0.0342
0.5658±0.0327
K1b (mL/μmol)
49.75±1.68
54.35±2.30
78.85±3.21
n1c
0.87±0.02
0.83±0.03
0.87±0.03
RMSE1d
0.128
0.179
0.072 14
qm2 a (mg/g gel)
43.12±5.33
43.69±4.12
45.33±4.23
K2 b (mL/mg)
0.078±0.008
0.144±0.009
0.205±0.012
qm2 a (μmol/g gel)
0.6465±0.0012
0.6550±0.0022
0.6796±0.0634
K2 b (mL/μmol)
5.20±0.53
9.60±0.60
13.67±0.80
n2 c
0.86±0.03
0.86±0.02
0.84±0.02
0.222
0.306
0.133
RMSE2
d
a
qm1 and qm2: saturation adsorption capacities of IgG and BSA, respectively;
b
K1 and K2: average association constants of IgG and BSA, respectively;
c
n1 and n2: heterogeneity parameters of IgG and BSA, respectively.
d
RMSE1 and RMSE2: root mean square error of adsorption isotherms of IgG and BSA,
respectively. The possible reasons for competitive adsorption between IgG and BSA on MMI resins can be attributed to the difference of partial hydrophobicity on the protein surface and molecular size. As shown with molecular simulation [7, 8, 33], there are some hydrophobic pockets on the surface of IgG, which might be more suitable for the binding to hydrophobic ligands such as MMI. Therefore, a stronger attraction might exist between IgG and MMI ligands due to the complicated hydrophobic interactions when comparing to BSA. Meanwhile, BSA with smaller molecular size, higher concentration and faster mobility can transfer and be adsorbed onto binding sites faster than IgG. However, IgG molecules would compete for the limited binding sites due to stronger protein-ligand attraction between IgG and HCIC ligands [29], which leads to the competitive adsorption behaviors.
15
3.2 Comparison of different resins As listed in Table 1, the qm value of IgG with MMI-B-4FF-100 resin was the highest among three resins tested, while that of BSA were similar, which suggests that MMI-B-4FF-100 exhibits the best binding ability of IgG and adsorption selectivity of IgG to BSA. To further evaluate the co-adsorption behaviors of IgG and BSA in binary systems, the molar ratios of IgG/BSA on MMI resins and those in the liquid phase were compared, and the results are shown in Fig. 4. It could be found that the molar ratios of IgG/BSA in the stationary phase were more than 5 times higher than those in the liquid phase, which indicates that IgG was more favorably bound to the MMI resins than BSA. For MMI-B-4FF-100, even 10 times of IgG/BSA molar ratios in the stationary phase than those in the liquid phase could be obtained. The results indicate that among the three MMI resins tested, MMI-B-4FF-100 possessed the most excellent adsorption ability as well as preferential adsorption of IgG to BSA. In addition, a linear relation (y=k*x) could be found between the molar ratios of IgG/BSA in the stationary phase and those in liquid phase, which could be used to describe and compare the preferential adsorption behavior of IgG to BSA. The correlated values of k and R2 are listed in Table 2. The k value of MMI-B-4FF-100 was the highest, which reveals the outstanding adsorption selectively of IgG to BSA. Fig. 4
Table 2. Parameters of linear fitting between the molar ratio of IgG/BSA in the stationary phase and in liquid phase with different MMI resins 16
a
MMI resins
MMI-B-3.5HF-50
MMI-B-4FF-100
MMI-B-6FF-110
ka
5.38
8.18
4.74
R2 b
0.9774
0.9403
0.9693
k means the slope of a linear relation between the molar ratios of IgG/BSA in the
stationary phase and those in liquid phase; b
R2 means the fitting degree of linear relation.
The results indicate that MMI-B-4FF-100 seems to be the most satisfactory resin to separate IgG selectively. The possible reasons are described as follows. As reported in our previous work [30], high ligand density on the internal surface of the MMI resin is necessary to provide enough attractive interactions to capture the protein. The pore surface ligand density (PSLD, ligand number/nm2) was proposed in our previous work [34], which is based on the hypothesis that the internal pore of resin can be regarded as idealized cylinders and the ligand distribute evenly on the internal pore [35]. The PSLD values of the three MMI resins tested were calculated as 0.77, 1.36 and 0.89 ligand number/nm2, respectively. It could be found that the PSLD value of the MMI-B-4FF-100 resin was the largest, which could provide higher accessibility and stronger hydrophobic interactions for IgG binding. Meanwhile, BSA was smaller and less hydrophobic than IgG, the attraction existed between BSA and MMI resin was weaker so the influence of PSLD on mass transfer and adsorption of BSA with MMI resin was less important. In general, among three MMI resins tested in the present work, MMI-B-4FF-100 showed the best adsorption selectivity of IgG to BSA 17
in the IgG/BSA binary adsorption, thus this resin would be the most suitable resin for the purification of IgG from the BSA-containing feedstock.
3.3 Protein adsorption behaviors in the column Protein adsorption on HCIC resins is typically pH-dependent [27, 36], and the control of pH is an effective way to adjust the adsorption of IgG and BSA on the MMI resins. Therefore, the adsorption behaviors of IgG and BSA in the column with MMI-B-4FF-100 resin were determined further at varying pHs to explore the optimal conditions for IgG separation from BSA-containing feedstock. The chromatographic results are shown in Fig. 5, and the adsorption proportions and the relative selectivity at different pH conditions are compared in Fig. 6. Fig. 5 Fig. 6 As shown in Fig. 6, remarkable difference could be found for IgG and BSA adsorption in the column with MMI-B-4FF-100 at varying pHs. For BSA, the adsorption amount increased sharply from 10.02% to 41.11 % as the pH increased from 4 to 5, and then decreased slightly at the pH range of 5~6, which further decreased to 3.72% with the increase of pH to 7. Compared with BSA, IgG kept high adsorption proportion at a relative wide range of pH 6~8, while adsorption reduced gradually as the pH changed from 6 to 4 or from 8 to 8.9. It could be found that IgG was adsorbed efficiently with the adsorption proportion of ~100% at pH 7, while BSA 18
was partially adsorbed with a maximum adsorption proportion of about 40% at pH 5.0. The pIs of IgG and BSA were around 6.8~10 and 4.8, respectively, and both IgG and BSA reached the maximum adsorption at the pHs around their individual pI. This phenomenon was consistent with that reported by Tong et al [27] and Yan et al [36]. Generally, protein molecule has relative small hydrodynamic diameter around the pI due to few net charges and thin hydrated layer on the surface, which would be favorable for the protein adsorption on hydrophobic interaction-based resins, such as MMI resins used in the present work [30]. The results of protein adsorption behaviors in the column indicate that pH control is a good way to improve IgG separation efficiency from binary IgG/BSA protein mixtures. At pH 7.0, almost all IgG was adsorbed on MMI-B-4FF-100 and most BSA was washed out, which indicates that IgG could be adsorbed selectively from the IgG/BSA protein mixtures. Meanwhile, near 100% IgG was found in non-adsorbed flowthrough fraction at pH 4.0 due to strong electrostatic repulsion between the positively-charged proteins and MMI ligands at acid condition. Therefore, the separation process of IgG from BSA-containing feedstock with MMI resin could be set as the adsorption-elution mode: equilibrium and loading at pH 7.0 to ensure the adsorption of IgG and the flowthrough of BSA, and then elution at pH 4.0 to obtain high recovery of IgG.
3.4 IgG separation from IgG/BSA protein mixture with MMI resins 19
The MMI-B-4FF-100 resin was used to separate IgG from BSA-containing feedstock under optimized separation conditions (loading at pH 7.0 and elution at pH 4.0). A mixture of IgG (1 mg/mL) and BSA (4 mg/mL) was used as a model feedstock to mimic the composition in serum. The chromatograms at various loading volumes (1, 2, 5, 10 mL) are shown in Fig. 7. The flowthrough and elution factions were collected and analyzed by SDS-PAGE and SEC-HPLC. The results of SDS-PAGE are also shown in Fig. 7. It can be found that the main compound in flowthrough fraction was BSA with few IgG detected, which indicates that IgG could be well adsorbed with MMI-B-4FF-100 resin at pH 7.0. Meanwhile, almost no BSA was detected in the elution fraction, which demonstrates that IgG could be separated efficiently from the binary IgG/BSA protein mixtures under the present separation condition. Fig. 7 The purities and recoveries of IgG at various loading volumes were determined by SEC-HPLC. For all the separation processes at various loading volumes, no BSA but only IgG monomer and few IgG aggregate were detected in the elution fractions. The purities of IgG monomer were above 95%, and the recovery of IgG monomer kept around 85-90% at various loading volumes. The results demonstrate that the MMI-B-4FF-100 resin can efficiently separate IgG from binary IgG/BSA protein mixtures. Compared to similar separation process with the commercial HCIC resin MEP-HyperCel reported by Tong et al [14, 27] (the purity of IgG was 62.9%), the 20
present process with the MMI resin was more efficient. For MEP-HyperCel resin, some additives like NaCl [27] or caprylate sodium [14] should be added in the loading buffer or washing buffer to improve IgG purity to reach about ~95%. Therefore, the MMI-B-4FF-100 resin with high ligand density and moderate pore size would be a promising HCIC resin for IgG purification. The results indicate that the combination of three factors, ligand density, pore size and hydrophobicity of ligand, determine together the separation performance of HCIC resins.
4. Conclusions In the present work, competitive adsorption behaviors between IgG and BSA were investigated with three MMI resins to study adsorption mechanism and enhance separation efficiency of IgG from the BSA-containing feedstock. The adsorption isotherms of single component and binary IgG/BSA protein mixtures at various mass ratios were measured, and the Langmuir-Freundlich adsorption model was used to fit the experimental data. The results showed that IgG could be adsorbed preferentially by MMI resins, and MMI-B-4FF-100 with moderate pore size and high ligand density exhibited the best adsorption selectivity of IgG over BSA. In addition, the binding behaviors of IgG and BSA in the column with MMI-B-4FF-100 were studied at various pHs to determine the optimal IgG separation conditions. Finally, IgG was separated from IgG/BSA protein mixtures using MMI-B-4FF-100, and high purity and recovery of IgG were obtained. The results indicated that suitable pore size and 21
ligand density of resins, and the control of pH are three critical factors to adjust the binding selectivity of IgG and improve separation efficiency of HCIC processes. MMI-B-4FF-100 with high ligand density and moderate pore size would be a promising HCIC resin for IgG purification.
Acknowledgments This work was supported by the National Natural Science Foundation of China and the China Postdoctoral Science Foundation (512100-X91603).
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Figure Captions Fig. 1 Adsorption isotherms of single and protein mixtures on MMI-B-3.5HF-50. (a) IgG; (b) BSA Fig. 2 Adsorption isotherms of single and protein mixtures on MMI-B-4FF-100. (a) IgG; (b) BSA Fig. 3 Adsorption isotherms of single and protein mixtures on MMI-B-6FF-110. (a) IgG; (b) BSA Fig. 4 Comparison of molar ratios of IgG and BSA on the resins and in liquid phase Fig. 5 Chromatograms of IgG (a) and BSA (b) in the column with MMI-B-4FF-100 resin at various pHs. For all curves, the first peak at about 5~10 min was the flowthrough peak, and the second peak at about 25 min was the CIP peak. Fig. 6 IgG and BSA adsorption in the column with MMI-B-4FF-100 resin as the function of loading pH Fig. 7 Chromatograms of IgG separation from IgG/BSA mixtures in the column with MMI-B-4FF-100. Insert: SDS-PAGE analysis of the fractions. FT and EL mean the fractions from flowthrough and elution pools, respectively.
29