Adsorption of IgG and BSA on Two Chromatographic ResinsâPoly

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Adsorption of IgG and BSA on Two Chromatographic ResinsPoly(ethylenimine)-4FF Resin and Tetrapeptidepoly(ethylenimine)-4FF Resin Yu-Ming Fang, Dong-Qiang Lin, and Shan-Jing Yao*

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Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China ABSTRACT: Poly(ethylenimine) (PEI) as a long flexible chain has been well used in protein chromatography with the advantages of high spatial utilization and “chain delivery” effect. In this work, two resins, i.e., poly(ethylenimine)-4FF resin (PEI-4FF resin) and Ac-YFRH-PEI-4FF resin by grafting tetrapeptide to PEI-4FF resin, were prepared for the protein adsorption and chromatographic separation. The pH-dependent adsorption properties of PEI-4FF resin and Ac-YFRH-PEI-4FF resin were discovered by getting the data of adsorption capacities of bovine serum albumin (BSA) and immunoglobulin G (IgG) under different pH conditions and buffer systems. The results showed high binding capacities of BSA on PEI-4FF resins across a range of pH values (5.0−8.9) with almost very little adsorption of IgG over a pH range from 5.0 to 8.0, indicating that IgG could be separated by flow-through method using PEI4FF resin. Since PEI has the advantage of having many flexible branched chains and Ac-YFRH (Ac-Try-Phe-Arg-His) as a peptide ligand has the advantages of comparatively high stability and low immunogenicity for antibody adsorption, Ac-YFRH-PEI-4FF resins were prepared by using PEI as the spacer arm and Ac-YFRH as the peptide ligand. The isotherm adsorption of the Ac-YFRH-PEI-4FF resin showed that the maximum binding capacity of IgG (qm = 123.6 mg/g resin, Kd = 0.10 mg/mL) was far more than that of BSA (qm = 62.0 mg/g resin, Kd = 0.25 mg/mL) at pH 8.0. Meanwhile, this tetrapeptide resin also showed high binding capacity of BSA (qm = 131.0 mg/g resin, Kd = 0.05 mg/mL) and low binding capacity of IgG (qm = 2.0 mg/g resin, Kd = 0.01 mg/mL) at pH 5.0, indicating that the separation of BSA and IgG could be achieved with the big differences in adsorption behaviors.

1. INTRODUCTION Monoclonal antibodies (mAb), which could be used to ward off the virus cell invasion by activating and strengthening the immune system, have been widely used in the treatment of cancer, autoimmune disease, and inflammation.1−4 In recent years, with the rapid development of antibody engineering, the expression level of antibody has been greatly improved, which could even reach up to 10 g/L for mAb.5 However, with the difficulties of obtaining a high purity of antibody, complications in the separation and purification process, and high cost of downstream processing techniques, it is important to find some new resins for antibody purification. In recent years, polymer-grafted chromatographic resins have been developed to improve the protein adsorption capacity and rates. Many polymers have been discovered, which could be well used to prepare resins, such as poly(ethylene glycol),6 dextran,7,8 poly(vinyl alcohol),9 and so on. Our group has done a lot of work in this area; Liu et al.10,11 have prepared new HCIC resins (MMI-B-XL) with dextran-grafted agarose gel as the matrix and 2-mercapto-1methyl-imidazole (MMI) as the functional ligand. The results indicated that these new HCIC resins with high ligand © XXXX American Chemical Society

densities were preferred for antibody purification by the stronger mixed-mode interactions and potential “chain delivery” effect. And compared to the nongrafted resins, the dextran-grafted layer on the resin could increase the ligand density, enhance the mass transport in the pore, and improve the dynamic adsorption at high kinetics. In the further research, Liu et al.12 have prepared new poly(glycidyl methacrylate)-grafted hydrophobic charge-induction resins with 5-aminobenzimidazole as the functional ligand. The results showed that the resins with the ligand density of 330 μmol/g (G-ABI-330 resin) had the maximum adsorption capacity and binding affinity for hIgG, which were better than the HCIC resins reported in the literature.13,14 Moreover, the dynamic binding capacity value at 10% breakthrough of the GABI-330 resins could reach to 76.3 mg/g at linear velocity 300 cm/h, which was also higher than those of nongrafted resins and other polymer-grafted HCIC resin (G-MMI and MMI-BXL-200).13,14 It is obvious that suitable polymers grafted to the Received: June 26, 2018 Accepted: October 17, 2018

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DOI: 10.1021/acs.jced.8b00535 J. Chem. Eng. Data XXXX, XXX, XXX−XXX

Journal of Chemical & Engineering Data

Article

advantages of Ac-YFRH and PEI chain in the further research.10 To get the further information on PEI used for antibody purification, both PEI-4FF resin and Ac-YFRH-PEI-4FF resin have been prepared in this study, and the adsorption binding capacities of IgG and BSA on these resins were evaluated. Based on the differences in adsorption behaviors to BSA and IgG, it is expected to present binding ability of new resins in the present work and provide some insights into the future development for antibody purification.

resins could improve the binding ability between chromatographic resins and antibody. Therefore, it is necessary to find a new polymer for the research of antibody adsorption. Poly(ethylenimine) (PEI) as a polymer molecule with positive charge has been found to be able to prepare anion exchange resins for protein purification.15 As macromolecules with long flexible chains, PEI could form an extended 3D adsorption space to enhance the binding ability of the resins.16 At the same time, the facilitated transport of adsorbed proteins, which was called the “chain delivery” effect, could be carried out by the chemical potential toward the bead center and the interactions of neighboring chains mediated by the bound molecules through the adjacent PEI chains.15,16 Therefore, using PEI is expected, which was grafted to Bestarose 4FF, to prepare the resin (PEI-4FF resin) for BSA and IgG adsorption with its powerful ion exchange. Subsequently, considering the electrostatic effect between PEI and IgG, it is necessary to couple something to PEI to shield part of the ion exchange function and increase affinity of the resin, which could reduce electrostatic effect and enhance hydrophobic effect for IgG adsorption. Further research was started by coupling peptide to PEI-4FF resin to extend its applications for antibody purification. Nowadays, biomimetic affinity chromatography, which was first proposed by Christopher R. Lowe in 1992, as a part of affinity chromatography, has been developed to adsorb and purify antibodies.17 Among many biomimetic affinity ligands, short peptide ligands have been successfully applied,18 which have raised interest in recent years as a means of antibody purification.19,20 In the previous work of our group, through molecular docking and molecular dynamics (MD) simulations, Wang et al.18 have discovered a tetrapeptide (AcYFRH, Ac-Try-Phe-Arg-His) ligand, which could be well used in IgG purification. The tetrapeptide resin was carried out by coupling Ac-YFRH to Bestarose 4FF agarose beads with hexamethylene diamine as spacer arm (Figure 1). The results

2. MATERIALS AND METHODS 2.1. Materials. Bestarose 4FF (4% highly cross-linked agarose beads) was purchased from Bestchrom Bio-Technology Co., Ltd. (Shanghai, China). The ligand of Ac-tyrosinephenylalanine-Argnine-Histidine (Ac-YFRH, purity: 97.6%) was synthesized by Chinese Peptide Company (Hangzhou, China). Human immunoglobulin G (hIgG) for intravenous injection was obtained from Boya Biopharmaceutical Ltd. (Jiangxi, China), and bovine serum albumin (BSA) was purchased from BBI Life Science (Shanghai, China). Poly(ethylenimine) (PEI) solution (average Mn ∼ 60 000 by GPC, average Mw ∼ 750 000 by LS, 50 wt % in H2O) was obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Allyl bromide (AB), N-bromosuccinimide (NBS), 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3- tetramethyluronium hexafluorophos-phate (HATU), N,N-diisopropylethylamine (DIPEA), and anhydrous dimethylformamide (DMF) were purchased from Aladdin Industrial Corporation (Shanghai, China). Other reagents were of analytical grade and used as received. 2.2. Preparation of PEI-4FF Resins and Ac-YFRH-PEI4FF Resins. PEI-4FF resin was prepared by grafting PEI onto Bestarose 4FF beads (4% highly cross-linked agarose beads) and the preparation process of the resin is shown in Figure 2. A total of 10.0 g of Bestarose 4FF agarose beads was drained and mixed with 10 mL of allyl bromide and 5 g of sodium hydroxide in 10 mL of 20% (v/v) dimethyl sulfoxide solution. The reaction was performed under 180 rpm and 30 °C for 24 h. Then, the beads were rinsed with ethanol and deionized water and added into 50% acetone solution with 5 g of NBS and reacted at 30 °C and 180 rpm for 1−3 h. The brominated beads were rinsed with deionized water and added into 1 M carbonate buffer (pH 10.0) to react with PEI (17.5 wt % in H2O) under 150 rpm and 30 °C for 12 h. The prepared resin was rinsed with deionized water, 0.1 M HCl, 0.1 M NaOH, and deionized water in sequence. Finally, the resin was drained and stored in 20% ethanol at 4 °C for further usage.18 The preparation of the Ac-YFRH-PEI-4FF resin, which is shown in Figure 2, could be carried out by coupling Ac-YFRH to PEI-4FF resin. A total of 3.0 g of PEI-4FF resin prepared before was sequentially rinsed with deionized water, ethanol, and DMF before peptide coupling. Subsequently, HATU and DIPEA were used to link peptide to the aminated resin. The molar ratio of peptide:amino groups:HATU:DIPEA was set as 1:1:2:4, and the reaction was performed under 170 rpm and 25 °C for 8 h. Then, the resin was washed extensively with DMF, ethanol, and deionized water. Before being stored in 20% ethanol at 4 °C, the resin was added into the mixture of acetic anhydride and sodium acetate at 25 °C for 1 h to block the untreated free primary amine on the resin to avoid unspecific adsorption.18 2.3. Determination of Amino and Ligand Density. The amino density (AD) of PEI-4FF resin was measured by

Figure 1. Structure of Ac-YFRH-4FF resin.

showed that Ac-YFRH resin could separate IgG from BSA containing feedstock with purity of 98.4% and recovery of 89.4% and purify mAb from CHO cell culture supernatant with purity of 99.5% and recovery of 84.2%. Obviously, the AcYFRH resin has several advantages to purify antibodies, such as keeping relatively high adsorption capacity over a broad range of pH 6.0−8.0 and salt concentration (0.0−1.0 M NaCl). Hence, Ac-YFRH as a peptide ligand was coupled to PEI-4FF resin to prepare a new peptide resin (Ac-YFRH-PEI-4FF resin), which could shield part of the ion exchange function of the PEI and increase affinity of the resin for IgG adsorption. While PEI as spacer arm could help ligands to increase the space efficiency by making them well distributed in the threedimensional (3D) pore spaces, it is excepted to use this resin for the separation of BSA and IgG by combining the B



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Figure 2. Preparation of the PEI-4FF resin and Ac-YFRH-PEI-4FF resin.

Zetasizer Nano ZS (Malvern Instruments, Malvern, Worcestershire, U.K.). The protein was dissolved in different buffer systems (acetate buffer for pH 4.0−5.0, phosphate buffer for pH 6.0−8.0, and Tris-HCl buffer for pH 8.9) to the concentration of 1 mg/mL. Then, each measurement was Carried out 15 times at 25 °C, and the results were obtained by average.21 Meanwhile, 0.1 g of resin was put in 1.5 mL of buffer (acetate buffer for pH 5.0, phosphate buffer for pH 6.0− 8.0, and Tris-HCl buffer for pH 8.9). Then, after the breaking of the resin by Glass Bead Breaker, each measurement was carried out 9 times at 25 °C, and the results were obtained by average. 2.5. Adsorption Isotherms. The IgG and BSA adsorption isotherms of the prepared PEI-4FF resin and Ac-YFRH-PEI4FF resin were measured in 20 mM acetate buffer (pH 5.0), 20 mM phosphate buffer (pH 6.0−8.0) and 20 mM Tris-HCl (pH 8.9) at 25 °C. A total of 30 mg of drained resins were mixed with 0.8 mL of IgG solution or BSA solution with different concentrations, and the mixture was incubated in a thermomixer under 1500 rpm and 25 °C for 3 h. The supernatant was separated by centrifugation at 7000 rpm, and unbound protein was measured with spectrophotometer (One Drop OD-1000+, Wins Technology, Nanjing, China) at 280 nm. The adsorption isotherm was described by the Langmuir equation (eq 2) as

the following steps. First, 4 g of drained gel was added to 40 mL of 2 M NaCl solution at 25 °C and 170 rpm for 30 min. Then, the resin was washed extensively with 100 mL of 0.1 M HCl and 500 mL of 0.0001 M HCl. After being drained, 1 g of PEI-4FF resin was used to react with 20 mL of Na2SO4 (10%, w/w) under 170 rpm and 25 °C for 2 h.16 Finally, the supernatant was collected by centrifuging at 8000 rpm for 1 min, and 0.05 M AgNO3 titration was carried out with K2CrO4 (5%, w/w) as the indicator to react with 10 mL of supernatant. The experiment was conducted in triplicate to get the ionic capacity of the resin calculated by mass balance. And the amino density was described by eq 1 as AD =

2 × 0.05 × V m

(1)

where AD is the amino density of the resin (mg/g resin), V is the consumed volume of the AgNO3 solution (mL), and m is the mass of the resin (mg), respectively. Subsequently, the ligand density of the Ac-YFRH-PEI-4FF resin can be calculated by the decrease of the peptide in the reaction solution. The peptide concentration was measured with reversed-phase high performance liquid chromatography (RP-HPLC) on Agilent 1100 series (Agilent Technologies, Santa Clara, CA). A Hypersil ODS-2 C18 column (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) was used. Solvent A was 0.01% TFA in H2O and solvent B was 0.09% TFA in the solution of 80% acetonitrile and 20% H2O. Ac-YFRH was analyzed using a stepwise gradient from 34% to 44% B in 20 min. UV detection of the tetrapeptide was performed at 220 nm. 2.4. Zeta Potential Measurements. The zeta potentials (ζ) of hIgG, BSA, and PEI-4FF resin were measured using the

q* =

qm × c* Kd + c*

(2)

where q* and c* are the equilibrium protein concentration on the resin (mg/g resin) and the equilibrium protein concentration in the solution (mg/mL), respectively. qm stands C



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for the saturation adsorption capacity (mg/g resin) and Kd is the dissociation constant (mg/mL).

3. RESULTS AND DISCUSSION 3.1. Preparation of PEI-4FF Resin and Ac-YFRH-PEI4FF Resin. After the determination of the amino density for the PEI-4FF resin, 7.6 mL of AgNO3 solution was consumed for titration. The results indicated that the amino density of the PEI-4FF resin was 760 μmol/g resin. Then, Ac-YFRH was coupled by HATU chemistry and the ligand density of the tetrapeptide resin was 260 μmol/g resin. To compare the surface morphologies among Bestarose 4FF, PEI-4FF resin, and Ac-YFRH-PEI-4FF resin, these resins were observed by scanning electron microscope (SEM) shown in Figure 3. The pictures showed that the reactions to gain the resins (PEI-4FF resin and Ac-YFRH-PEI-4FF resin) would not destroy the structure of the Bestarose 4FF. 3.2. Adsorption of BSA and hIgG with PEI-4FF Resin. The adsorption isotherm of PEI-4FF resin was measured using IgG and BSA as model proteins at pH 5.0−8.9 under 25 °C. Figure 4 shows the adsorption isotherm and corresponding Langmuir adsorption fitting results with maximum binding capacity (qm) and dissociation constant (Kd) values. The data were carried out by mixing 30 mg of PEI-4FF resin with 0.8 mL of IgG solutions or BSA solutions with different concentrations in the range of pH 5.0−8.9. For example, at pH 8.0, 30 mg of PEI-4FF resins were added into 0.8 mL of BSA solutions with the concentrations of 0.45 ± 0.00, 1.05 ± 0.00, 1.93 ± 0.00, 2.86 ± 0.01, 3.76 ± 0.00, 5.61 ± 0.01, 7.39 ± 0.01, and 9.4 ± 0.01 mg/mL. Then, after the reaction of the resin and the protein, BSA solutions with the concentrations of 0.01 ± 0.01, 0.03 ± 0.00, 0.04 ± 0.00, 0.14 ± 0.00, 1.76 ± 0.00, 3.25 ± 0.01, and 5.18 ± 0.00 mg/mL were detected, respectively. In this situation, the fitting equation of the 111.5 × c * Langmuir model is q* = 0.08 + c * . Then, the ability of the Langmuir in fitting experimental data is listed in Table 1 in terms of the absolute deviation, Δq = qcal − qexp

(3)

where Δq is absolute deviation (mg/g resin), qcal is the amount adsorbed gained by calculated (mg/g resin), and qexp is the amount adsorbed from experiments (mg/g resin). And the relative deviation was described by eq 4 as R=

Δq × 100% qexp

Figure 3. SEM images of (A) Bestarose 4FF, (B) PEI-4FF resin, and (C) Ac-YFRH-PEI-4FF resin.

BSA on the resin is caused by electrostatic interaction, enhancement of the shielding leads to the reduction of the binding ability (from 160.4 mg/g resin to 91.5 mg/g resin). At pH 6.0 to pH 8.0, as the adsorption is carried out in the same buffer system, the binding ability of the resin is only influenced by the change of pH value, which shows the rule that by the increase of the pH value, the adsorption ability of the resin also increases slowly via the enhancement of the electrostatic interaction (from 91.5 mg/g resin to 111.5 mg/g resin). At pH 8.0 to pH 8.9, rapid growth of the binding ability with the buffer system changes from phosphate buffer to Tris-HCl solution, indicating that the reduction in shielding could strengthen the binding ability between BSA and the resin (from 111.5 mg/g resin to 182.0 mg/g resin). The pIs of IgG and BSA were measured at pH 6.5 and 4.8 as shown in Figure 5, respectively, and the zeta potentials of the PEI-4FF resin are seen in Figure 6; the result that the ability of PEI-4FF resin to

(4)

where R is the relative deviation, Δq is absolute deviation (mg/ g resin), and qexp is the amount adsorbed from experiments (mg/g resin). The relative deviation for the PEI-4FF resin to BSA at pH 8.0 is also shown in Table 1. It could be seen from the Table 1 that the adsorption data conformed to the isotherm of the Langmuir adsorption; therefore, the sorption isotherms were modeled using the Langmuir equation (eq 2) shown in Figure 4. The results in Figure 4A show that the binding ability of the PEI-4FF resin to BSA follows the rule of falling first and then rising, which is caused by the changes of the buffer systems and pH values. At pH 5.0 to pH 6.0, the buffer system changes from acetate buffer to phosphate buffer adding the shielding of the amino effect to the resin with the increase of the ionic binding capacity by the buffer system. While the binding capacity of the D



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Figure 5. Zeta potentials of IgG and BSA.

Figure 6. Zeta potentials of PEI-4FF resin.

Figure 4. Adsorption isotherms of (A) BSA and (B) IgG on PEI-4FF resin.

Table 2. Equilibrium Parameters qm and Kd of PEI-4FF Resin for Protein Adsorption at Different pH Values

Table 1. Deviations of the Calculated Adsorbed Amount and the Experimental Values of the BSA on the PEI-4FF Resins at pH 8.0 equilibrium concentration (mg/mL)

qexp (mg/g)

qcal (mg/g)

|Δq| (mg/g)

|R|

0.01 0.03 0.04 0.43 1.76 3.25 5.18

11.51 27.11 49.74 89.10 103.70 109.67 114.05

12.39 30.41 37.17 94.00 106.65 108.82 109.80

0.88 3.3 12.57 4.9 2.95 0.85 4.25 4.24

7% 12% 25% 5% 3% 1% 4% 8%

average

BSA

pH pH pH pH pH

5.0 6.0 7.0 8.0 8.9

qm (mg/g)

Kd (mg/mL)

160.4 91.5 99.0 111.5 182.0

0.05 0.04 0.07 0.08 0.05

IgG R2

qm (mg/g)

Kd (mg/mL)

R2

0.96 0.94 0.96 0.98 0.96

10.0 8.0 32.0 20.0 210.0

6.40 6.30 13.00 10.00 10.00

0.99

3.3. Adsorption of BSA and IgG with Ac-YFRH-PEI4FF Resin. The adsorption isotherm of Ac-YFRH-PEI-4FF resin was also measured using IgG and BSA as model proteins at pH 5.0−8.9 under 25 °C. Figure 7 shows adsorption isotherm and corresponding Langmuir adsorption fitting results with maximum binding capacity (qm) and dissociation constant (Kd) values. The data were also carried out by mixing 30 mg of PEI-4FF resin with 0.8 mL of IgG solutions or BSA solutions with different concentrations in the range of pH 5.0− 8.9. For example, at pH 8.0, 30 mg of Ac-YFRH-PEI-4FF resins were added into 0.8 mL of IgG solutions with the concentrations of 0.33 ± 0.01, 0.71 ± 0.01, 1.49 ± 0.01, 2.91 ± 0.01, 4.42 ± 0.02, 5.92 ± 0.00, and 7.38 ± 0.01 mg/mL. Then,

adsorb BSA is far greater than the ability to adsorb IgG at pH 5.0 could be well explained by the electrostatic repulsion between PEI and IgG. Then, the different binding abilities could lead to the separation of IgG and BSA by binding BSA and flowing through IgG at pH 5.0. Meanwhile, Table 2 shows qm and Kd values of PEI-4FF resin to BSA and IgG, which are calculated by the Langmuir equation (eq 2). All the Kd values of the BSA on the resins shown in Table 2 are described by the Langmuir model resin to BSA. E



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Table 3. Deviations of the Calculated Adsorbed Amount and the Experimental Values of the IgG on the Ac-YFRHPEI-4FF Resins at pH 8.0 equilibrium concentration (mg/mL)

qexp (mg/g)

qcal (mg/g)

|Δq| (mg/g)

|R|

0.01 0.02 0.03 0.20 0.46 1.62 2.91

8.51 18.71 38.67 71.79 104.90 115.82 120.81

11.23 20.60 28.52 82.40 101.53 116.41 119.49

2.72 1.89 10.15 10.61 3.37 0.59 1.32 4.38

32% 10% 26% 15% 3% 1% 1% 12%

average

Table 4. Equilibrium Parameters qm and Kd of Ac-YFRHPEI-4FF Resin for Protein Adsorption at Different pH Values BSA

pH pH pH pH pH

5.0 6.0 7.0 8.0 8.9

IgG

qm (mg/g)

Kd (mg/mL)

R2

qm (mg/g)

Kd (mg/mL)

R2

131.0 95.3 95.6 62.0 13.2

0.05 0.07 0.11 0.25 0.62

0.94 0.97 0.95 0.97 0.77

2.0 90.2 114.2 123.6 20.0

0.01 3.16 0.46 0.10 0.02

0.99 0.99 0.98 0.96

capacities of the peptide resin to BSA and IgG at pH 8.9 had dropped a lot compared to the adsorption capacities at pH 8.0, which was not shown in the PEI-4FF resin, indicating that the graft of short peptide could influence the endurance of PEI to the change of pH. In addition, the Ac-YFRH-PEI-4FF resin has higher ability to adsorb IgG (123.6 mg/g resin) than BSA (62.0 mg/g resin) at pH 8.0, while it could be easier to adsorb BSA (131.0 mg/g resin) than IgG (2.0 mg/g resin) at pH 5.0. Hence, further research of IgG separation could be carried out by absorbing at pH 8.0 and eluting at pH 5.0. Table 4 shows qm and Kd values of Ac-YFRH-PEI-4FF resins to BSA and IgG calculated by the Langmuir equation (eq 2). Compared to the highest adsorption ability of IgG on Ac-YFRH resin reported before (qm = 100.5 mg/g resin, pH 8.0), Ac-YFRH-PEI-4FF resin (qm = 123.6 mg/g resin, pH 8.0) used PEI as spacer arm has certain comparability for IgG adsorption.18 Moreover, the Kd value of the IgG on the Ac-YFRH-PEI-4FF resin at pH 8.0 is 0.1 mg/mL, which is absolutely less than the Kd values reported of Ac-YFRH resin,18 indicating that PEI could enhance the affinity of the resin by increasing space utilization.

Figure 7. Adsorption isotherms of (A) BSA and (B) IgG on AcYFRH-PEI-4FF resin.

after the reaction of the resin and the protein, the IgG solutions with the concentrations of 0.01 ± 0.01, 0.02 ± 0.00, 0.03 ± 0.00, 0.20 ± 0.00, 0.46 ± 0.01, 1.62 ± 0.01, and 2.91 ± 0.02 mg/mL were detected, respectively. The fitting equation 123.6 × c * of this situation by the Langmuir model is q* = 0.10 + c * . The deviation of the calculated adsorbed amount and the experimental values of the IgG on the Ac-YFRH-PEI-4FF resins at pH 8.0 are shown in Table 3 carried out by eq 3 and eq 4. The adsorbing data conformed to the isotherm of Langmuir adsorption; therefore, the sorption isotherms on the Ac-YFRHPEI-4FF resins shown in Figure 7 were also correlated using the Langmuir equation (eq 2). In Figure 7, it is obvious that, compared to the PEI-4FF resin, shielding part of the ion exchange function of the PEI and increasing affinity of the resin could reduce the binding ability of BSA and strengthen the binding ability of IgG at pH 5.0−8.0. Moreover, the adsorption of IgG by the peptide resin shows strong pHdependency which could be seen in Table 4. When pH changed to 8.9, qm dropped significantly to 20.0 mg/g resin. At pH 5.0−8.0, the increase of qm with the increase of pH was due to the change of the electrostatic potential surface of the Fc fragment.18 Then, it could be found that the adsorption

4. CONCLUSIONS In this work, two kinds of resins, PEI-4FF resin and Ac-YFRHPEI-4FF resin, were prepared for the adsorption of BSA and IgG under different pH and buffer systems. The results showed that high adsorption capacity of BSA and low adsorption capacity of IgG could be carried out by PEI-4FF resin at pH 5.0, indicating that IgG purification could be achieved by flowthrough method. Further research showed that the Ac-YFRHPEI-4FF resin could obtain high adsorption capacity of IgG and low adsorption capacity of BSA at pH 8.0, while high adsorption capacity of BSA and low adsorption capacity of IgG could be achieved at pH 5.0, indicating that IgG separation on the new peptide resins could be expected by adsorbing IgG at pH 8.0 and eluting it at pH 5.0. F



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with 5-aminobenzimidazole as a functional ligand. J. Sep Sci. 2016, 39, 3130−3136. (13) Liu, T.; Lin, D.-Q.; Wu, Q.-C.; Zhang, Q.-L.; Wang, C.-X.; Yao, S.-J. A novel polymer-grafted hydrophobic charge-induction chromatographic resin for enhancing protein adsorption capacity. Chem. Eng. J. 2016, 304, 251−258. (14) Tong, H.-F.; Lin, D.-Q.; Chu, W.-N.; Zhang, Q.-L.; Gao, D.; Wang, R.-Z.; Yao, S.-J. Multimodal charge-induction chromatography for antibody purification. J. Chromatogr A 2016, 1429, 258−264. (15) Zhao, Y. Y.; Dong, X. Y.; Yu, L. L.; Liu, Y.; Sun, Y. Characterization of new polymer-grafted protein cation exchangers developed by partial neutralization of carboxyl groups derivatized by modification of poly(ethylenimine)-Sepharose with succinic anhydride. J. Chromatogr A 2018, 1550, 28−34. (16) Yu, L. L.; Tao, S. P.; Dong, X. Y.; Sun, Y. Protein adsorption to poly(ethylenimine)-modified Sepharose FF: I. A critical ionic capacity for drastically enhanced capacity and uptake kinetics. J. Chromatogr A 2013, 1305, 76−84. (17) Burton, N. P.; Lowe, C. R. Design of novel affinity adsorbents for the purification of trypsin-like proteases. J. Mol. Recognit. 1992, 5, 55−68. (18) Wang, R.-Z.; Lin, D.-Q.; Chu, W.-N.; Zhang, Q.-L.; Yao, S.-J. New tetrapeptide ligands designed for antibody purification with biomimetic chromatography: Molecular simulation and experimental validation. Biochem. Eng. J. 2016, 114, 191−201. (19) Cserhati, T. Chromatography of amino acids and short peptides. New advances. Biomed. Chromatogr. 2007, 21, 780−796. (20) Fang, Y.-M.; Lin, D.-Q.; Yao, S.-J. Review on biomimetic affinity chromatography with short peptide ligands and its application to protein purification. J. Chromatogr A 2018, 1571, 1. (21) Tong, H.-F.; Lin, D.-Q.; Yuan, X.-M.; Yao, S.-J. Enhancing IgG purification from serum albumin containing feedstock with hydrophobic charge-induction chromatography. J. Chromatogr A 2012, 1244, 116−122.

In general, this work has demonstrated that PEI could be well used for the protein adsorption and it could be the new direction for peptide resin to use PEI as spacer arm with several advantages, such as increasing space utilization.

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AUTHOR INFORMATION

Corresponding Author

*Shan-Jing Yao. Fax: +86-571-87951982. E-mail: yaosj@zju. edu.cn. ORCID

Dong-Qiang Lin: 0000-0002-0504-8391 Shan-Jing Yao: 0000-0003-3199-3044 Funding

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

The authors declare no competing financial interest.

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REFERENCES

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