A novel polymer-grafted hydrophobic charge-induction chromatographic resin for enhancing protein adsorption capacity

Chemical Engineering Journal 304 (2016) 251–258

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A novel polymer-grafted hydrophobic charge-induction chromatographic resin for enhancing protein adsorption capacity Tao Liu, Dong-Qiang Lin ⇑, Qi-Ci Wu, Qi-Lei Zhang, Cun-Xiang Wang, Shan-Jing Yao ⇑ Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Poly(GMA)-grafted HCIC resins were

prepared with controlled grafting density and ligand density.
AGET ATRP was used for the controlled preparation of polymergrafted resins.
Adsorption capacity and pore diffusion reached maximum at intermediate ligand density.
Dynamic binding capacities of IgG were improved with optimized G-MMI resins.
Good performance of G-MMI resin was verified by IgG separation for 50 cycles.

a r t i c l e

i n f o

Article history: Received 1 March 2016 Received in revised form 11 June 2016 Accepted 13 June 2016 Available online 14 June 2016 Keywords: Polymer-grafted resin Hydrophobic charge-induction chromatography Ligand density Grafting density Protein adsorption

a b s t r a c t Hydrophobic charge-induction chromatography (HCIC) is a developing technology for antibody purification. To enhance the protein adsorption capacity, a novel polymer-grafted HCIC resin was developed, in which the surface-initiated activator generated by electron transfer (AGET) atom transfer radical polymerization (ATRP) was explored as a controlled polymerization technique to reconstruct matrix structure and ligand distribution. Using poly(glycidyl methacrylate, GMA) as grafting polymer and 2-mercapto-1methyl-imidazole (MMI) as functional ligand, poly(GMA)-grafted HCIC resins were prepared with series of grafting and ligand densities. Adsorption behaviors of human immunoglobulin G (hIgG) on the prepared resins demonstrated the necessity of controlling grafting and ligand density. Saturated adsorption capacity (Qm) and effective pore diffusivity (De) reached the maximum under medium ligand density when the grafting density was kept constant. The highest Qm and De values were found under the highest grafting density, which were 73% and 7.17 times higher than the non-grafted resin, respectively. Column breakthrough tests indicated that the dynamic binding capacity of the resin with optimized grafting density and ligand density was up to 34.6 mg/g when linear velocity was 300 cm/h, which was 86.3% higher than dextran-grafted resin. The resin was then used to separate hIgG from a protein mixture (hIgG/ human serum albumin = 1:4), high purity (>99%) and recovery (>90%) of hIgG were found with 50cycle reuses, which verified the selectivity and robustness of G-MMI resin prepared. In general, the surface-initiated AGET ATRP provides a controlled grafting strategy to improve protein binding capacity for chromatographic separation, and new resins developed have great potential in large-scale protein purification applications. Ó 2016 Published by Elsevier B.V.

⇑ Corresponding authors. E-mail addresses: [email protected] (D.-Q. Lin), [email protected] (S.-J. Yao). http://dx.doi.org/10.1016/j.cej.2016.06.074 1385-8947/Ó 2016 Published by Elsevier B.V.

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1. Introduction Hydrophobic charge-induction chromatography (HCIC) is a new technology for antibody capture, and its effectiveness has been demonstrated by many successful applications due to good adsorption selectivity, salt-tolerance property and milder elution condition [1–3]. However, because of relatively weak hydrophobic interactions between HCIC ligands and antibodies, the dynamic binding capacities (DBC) decreased sharply with the increasing column throughout. For example, DBC of immunoglobulin Y (IgY) with HCIC resin Streamline-MMI was only 1.3 mg/mL [4], and it was only 3.2–3.5 mg/mL for serotype B fragment with MEP HyperCel resin [5]. Therefore, HCIC resins still need to be improved for large-scale industrial applications. Polymer grafting on intrapore surface of resin beads is an effective strategy to improve protein binding capacity. The grafted polymer chains can help functional ligands fill the porous volume in the intrapore, which provides a three dimensional scaffold in protein binding [6–14]. Dextran-grafted resins are one of most typical polymer-grafted chromatographic resins [6,15], which were prepared by ‘‘grafting to” methods [16], i.e. dextran chains were grafted directly on the intrapore surface of resin beads [6,7]. However, there are some drawbacks of this ‘‘grafting to” method. Firstly, these polymer-grafted resins were prepared by coupling the soft inclusion (dextran) onto the pore surface of resin beads directly, which makes grafting density cannot be well controlled and characterized. Thus the effects of grafting density on the protein adsorption and kinetics cannot be optimized [9]. Secondly, the grafting density would be limited due to relatively slow diffusion kinetics and steric hindrance of the attached macromolecules such as dextran [17]. Finally, for dextran-grafted resins, ligands exist both in grafting layers and on pore surfaces, which cause an obvious heterogeneity of ligand distribution. Another type of polymer-grafted chromatographic resin was developed by the ‘‘grafting from” method, which uses in situ polymerization to form grafting polymer on the intrapore surface of resin beads [18]. Recently, surface-initiated atom transfer radical polymerization (SI-ATRP) was developed as one of ‘‘grafting from” methods to form polymer chains with uniform molecular mass distribution, which could control grafting density and grafting length independently on the surface of membranes by adjusting initiator and monomer dosages [19–21]. As reported by Lamprou et al. [22], ATRP was used to prepare the temperature-responsive polymer-grafted chromatographic resins. In ATRP reaction, the radicals could be formed through a reversible redox process catalyzed by a transition metal complex, which would generate the oxidized metal complexes as persistent radicals to reduce the stationary concentration of growing radicals and thereby minimize the contribution of termination [23]. Recently the amount of catalysts has been reduced significantly for ATRP [24–26], but a strict oxygen-free environment is still needed to initiate the ATRP process. The surface-initiated activator generated by electron transfer (AGET) could form the active catalyst Cu(I) continuously in situ by adding reducing agents. Thus, surface-initiated AGET combined with ATRP would not need any de-oxygenation operation [27,28]. Unfortunately, AGET ATRP has not been used for the preparation of polymer-grafted chromatographic resins till now. In the present work, the surface-initiated AGET ATRP was explored to construct new polymer-grafted agarose resins for HCIC with highly controlled grafting. Agarose gel was used as the matrix with small pore size (43.2 nm) [29], and poly(glycidyl methacrylate, GMA) was used as the grafted layer with 2-mercapto-1-methyl-imidazole (MMI) as the functional ligand. Poly(GMA)-grafted HCIC resins G-MMI with different grafting densities and ligand densities were prepared. The adsorption equilibrium, kinetics and DBCs of human immunoglobulin G (hIgG) onto series of G-MMI resins with different grafting and ligand

densities were investigated and compared with that of nongrafted, dextran-grafted and commercialized HCIC resins. The mechanism on the cross-effect of grafting density and ligand density on protein adsorption was discussed. Finally, the separation behaviors of G-MMI resins for the protein mixture (hIgG/human serum albumin = 1:4) and DBCs of hIgG onto G-MMI resins during the reusing cycles were tested. 2. Experimental 2.1. Materials 4% cross-linked agarose gel (Bestarose 4FF) was purchased from Bestchrom Bio-Technology Co., Ltd. (Shanghai, China). MEP HyperCel was purchased from Pall Corporation (East Hills, NY, USA). Human c-globulin (human normal immunoglobulin G, IgG > 98%) was obtained from Merck KGaA (Darmstadt, Germany). hIgG for intravenous injection (IgG > 96%) was purchased from RAAS Blood Products Co., Ltd. (Shanghai, China). Human serum albumin (HSA > 99%) was purchased from Sigma (Milwaukee, WI, USA). Ascorbic acid (99%), 2-bromoisobutyryl bromide (2-BIB, 98%), copper(II) bromide (CuBr2, 99.95%), 4-dimethylaminopyridine (DMAP, 99%), glycidyl methacrylate (GMA, 97%), 2-mercapto-1methylimidazole (MMI, 98%), N,N,N0 ,N00 ,N00 -pentamethyldiethylene triamine (PMDETA, 99%), 2-propanol (P99.8%), tetrahydrofuran (THF, anhydrous, P99.9%) and triethylamine (TEA, P99%) were purchased from Aladdin (Shanghai, China). Acetone (P99.5%), sodium carbonate (P99.8%), sodium bicarbonate (P99.8%), sodium hydroxide (P96%), sodium chloride (P99.5%), hydrochloric acid solution (36.0–38.0%), acetic acid (P99.5%), sodium acetate (P99%), disodium phosphate (P99%) and sodium phosphate dibasic (P99%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Preparation of polymer-grafted HCIC resins The preparation route of the polymer-grafted HCIC resins with the surface-initiated AGET ATRP is shown in Fig. 1. Typically, 2 g agarose gels were washed with 20 mL acetone for 1 h, and then added into a mixture of anhydrous THF (20 mL), acid-binding agent TEA (0.22–2.2 mL) and catalyst DMAP (25 mg). Different amounts of initiator precursors 2-BIB (0.2–2.0 mL) were added dropwise into the solution at 0 °C, and the molar ratio of 2-BIB to TEA was kept at 1:1. The solution was agitated at 0 °C for 2 h and then agitated continuously at 30 °C for 24 h for the esterification reaction, and the esterificated agarose gels were obtained. Then, monomer GMA (0.1–1.2 mL) was added into 25 mL 80% 2-propanol solution. A catalyst was formed in situ by adding CuBr2 (5 mg), PMDETA (10 mg), and reducing agent ascorbic acid (10 mg) to the solution. The solution was then added into a flask containing the activated agarose gels and reacted at 50 °C for 24 h. The poly(GMA)-grafted agarose gels were then thoroughly washed with acetone and deionized water. Finally, the poly(GMA)-grafted agarose gels were mixed with MMI ligands at a 1:1 molar ratio of MMI to GMA in 1 M carbonate buffer (pH 11) at 50 °C for 24 h, and G-MMI resins were obtained. 2.3. Determination of grafting density and ligand density of G-MMI resins Grafting density (lmol/g gel) was defined as the number of polymer chains onto the matrix surface. However, due to the difficulty on direct measurements of grafting density of resin beads, in the present work the grafting density was represented indirectly by the amount of initiator sites, corresponding to the consumption

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253

Fig. 1. Illustration of the controlled preparation of poly(GMA)-grafted HCIC resin G-MMI.

of 2-BIB during the reaction. So grafting density was calculated according to mass balance calculation of 2-BIB concentration in the solution [20], which was determined by HPLC (Agilent 1100 system) under UV absorbance at 220 nm with an organic acid analysis column (Bio-Rad Aminex HPX-87H ion-exchange column, 300 mm  7.8 mm). The mobile phase was 0.005 M H2SO4 with a flow rate of 0.5 mL/min. The column was kept at 40 °C. Sample injection volumes were 10 lL. G-MMI resin is a weak base resin, so the titration with HCl was used to measure the ligand density. The pKa of MMI ligand is 5.3. Based on the titration curve, pH 3.8 was chosen as the titration endpoint [2]. Specifically, 1 g drained G-MMI resin was washed with 0.1 M NaOH solution and deionized water, then drained and transferred to a vial. 5 mL 0.5 M NaCl solution was added into the vial, and the mixture was titrated with 0.1 M HCl solution to pH 3.8 [30]. The ligand density on G-MMI was calculated and expressed in lmol/g gel as follows,

D¼

Titer  0:1 Adsorbent

ð1Þ

where D is the ligand density on G-MMI resin, Titer is the titer of 0.1 M HCl solution (lL). Adsorbent is the mass of G-MMI resin (g).

containing different concentrations of hIgG (0.5–10 mg/mL) in 2 mL centrifuge tubes. The mixture was agitated in a thermomixer (1500 rpm) at 25 °C for 5 h to reach equilibrium. Then the resins were separated by centrifugation (4000g), and protein concentrations in the supernatant were determined at 280 nm with a spectrophotometer (One DropTM Spectrophotometer, Nanjing Wins Technology Co., Ltd., Nanjing, China). The equilibrium adsorption capacity of resin (mg/g gel), Q* was calculated based on the following equation,

Q ¼

ðC 0  C  Þ  V m

ð2Þ

where C0 is the initial protein concentration (mg/mL) before adsorption, C⁄ is the protein concentration in the liquid (mg/mL) when the adsorption reached equilibrium, m is the mass of drained resins (g), V is the liquid volume (mL). The adsorption isotherm was fitted by the Langmuir equation,

Q ¼

Q m  K  C 1 þ K  C

ð3Þ

where Q⁄ is the equilibrium adsorption capacity of resin (mg/g gel), Qm is the saturated adsorption capacity (mg/g gel) and K is the equilibrium constant (mL/mg).

2.4. Adsorption equilibrium 2.5. Adsorption kinetics Adsorption isotherms of hIgG on G-MMI resins with different grafting and ligand densities were determined by batch adsorption equilibrium experiments. Non-grafted MMI-L100 resin (ligand density 100 lmol/g) was tested as a control. 0.04 g drained resin was added to 0.8 mL 20 mM sodium phosphate buffer (pH 7.0)

Adsorption kinetics of hIgG on different G-MMI resins and MMIL100 were measured by a batch method. 2 g drained HCIC resins were added into 40 mL protein solution (2 mg/mL hIgG in 20 mM sodium phosphate buffer at pH 7.0) in a 50 mL flask. The resins

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were fully suspended in the flask by magnetical agitation at 25 °C at 280 rpm. The protein solution was pumped out and flowed through a 2 lm stainless filter. The real-time concentration of protein in the suspension was monitored by a UV monitor (UV Detector K-2600, Knauer, Germany). The adsorption kinetics curves were obtained and fitted by the pore diffusion model (PDM) using the Matlab 7.5.0 (Mathworks, USA) [31]. PDM is one of the most widely used models to describe the mass transfer for the adsorption of protein onto the porous resin. The effective pore diffusivity De was calculated based on the previous reported method [3]. 2.6. Column breakthrough Column breakthrough experiments for G95-MMI-L300, G140MMI-L445 and G170-MMI-L365 resins were performed with ÄKTAexplorer 100 system (GE Healthcare, Uppsala, Sweden). MMI-L100 and MEP HyperCel were also tested as two controls. A Tricorn column 5/100 (inner diameter 5 mm, length 100 mm) with 2 mL HCIC resin was used. hIgG for intravenous injection was used as the loading protein. All experiments were performed at 25 °C in 20 mM sodium phosphate buffer (pH 7.0). After the resins were packed into the column, the column was equilibrated with 10 column volumes of the equilibrium buffer until a stable baseline of absorbance at 280 nm was reached. Then 2.0 mg/mL hIgG solution prepared in 20 mM sodium phosphate buffer (pH 7.0) was loaded at linear velocities of 100, 200 and 300 cm/h. The protein concentration in the effluent was monitored by a UV monitor (UV Detector K-2600, Knauer, Germany). The path length of the detector was 10 mm, and the molar absorption coefficient was 2,100,000 L/(mol cm). Then the column was eluted with acetate buffer (pH 4.0), regenerated with 0.1 M NaOH and re-equilibrated with 20 mM sodium phosphate buffer (pH 7.0) in sequence. DBC at 10% breakthrough, Q10% (mg/g gel), was calculated as following,

Q 10% ¼

C0 

0

2.8. Determination of hIgG purity and recovery The analytical SEC-HPLC was performed with LC3000 HPLC system (Beijing ChuangXinTongHeng Science and Technology Co., Ltd., Beijing, China) using the TSK G3000SWXL column (7.8 mm  30.0 cm, TOSOH, Japan). The mobile phase of 0.2 M sodium phosphate buffer (pH 7.0, containing 1% isopropanol) was used after 0.22 lm membrane filtration and degassing. The operation flow rate was 0.6 mL/min. The HPLC purity of hIgG was defined as the percentage of the peak area of hIgG to the total integrated peak areas. The recovery of hIgG was calculated as the percentage of hIgG amount in the elution fraction to that in the feedstock. 2.9. Removal of host cell proteins and DNA from cell culture supernatant The separation of monoclonal antibody (mAb) and removal of host cell proteins (HCP) and DNA from Chinese hamster ovary (CHO) cell culture supernatant were performed on ÄKTAexplorer 100 system using Tricorn 5/50 column with 1.0 mL G170-MMIL365 resin. The experimental condition was similar as that in Section 2.7, loading at pH 7.0 and elution at pH 4.5. HCP content was determined using CHO HCP ELISA Kit 3 G F015 from Cygnus Technologies (Southport, NC, USA) [32]. DNA content was determined using a CFX96 TouchTM Real-Time PCR Detection Systems (Bio-Rad Laboratories) with the method in the literature [33]. 3. Results and discussions 3.1. Controlled preparation of poly(GMA)-grafted HCIC resins

R V 10%

ð1  C=C 0 ÞdV V s  qs

were tested after 30 and 50 cycles to validate the adsorption property of G-MMI resin.

ð4Þ

where C and C0 are the protein concentration of outlet fluid and initial fluid, respectively. V10% is the loading volume at 10% breakthrough and Vs is the settled volume of the gel. qs is the settled density of resin beads in the column (g drained gel/mL settled gel). The dead volume of flow path was subtracted to determine the loading volume.

Poly(GMA)-grafted HCIC resins G-MMI were prepared with three major steps (Fig. 1), i.e. gel esterification, grafting with AGET ATRP and ligand coupling. Firstly, the agarose gel was esterificated with 2-bromoisobutyryl bromide (2-BIB) to introduce the initiator onto the pore surface of agarose gel. The amount of initiator could be calculated based on the consumption of initiator precursor (2-BIB) in solution, which represented grafting density for further GMA grafting. As shown in Fig. 2, the grafting density could be controlled by adjusting 2-BIB addition in solution. The grafting density

2.7. hIgG separation from protein mixture and resin reusability test A protein mixture of 1 mg/mL hIgG and 4 mg/mL human serum albumin (HSA) was used as the mimic human serum to investigate the selectivity of G-MMI resin [32]. ÄKTAexplorer 100 (GE Healthcare, Uppsala, Sweden) was used. A Tricorn 5/50 column (inner diameter 5 mm, length 50 mm) (GE Healthcare, Uppsala, Sweden) was packed with 1.0 mL G170-MMI-L365 resin. The column was first equilibrated with 10 column volumes (CVs) of equilibration buffer (20 mmol/L sodium phosphate buffer (pH 7.0). 10 mL protein mixtures were then loaded and washed with 10 CVs of equilibration buffer to remove unbound proteins. The bound proteins were eluted with 20 mmol/L acetate buffers (pH 4.0, 4.5, 5.0 and 5.5) and the column was regenerated with 5 CVs of 0.1 mol/L NaOH. The flow rate was 1 mL/min. The chromatographic run was monitored online at 280 nm. The collected fractions were analyzed with SEC-HPLC. To assess the reusability of G-MMI resin, hIgG separation from the protein mixture was repeated 50 cycles with same procedure as mentioned above. The purity and recovery of hIgG were evaluated. In addition, DBC values at 10% breakthrough (Q10%) of hIgG

Fig. 2. Grafting density of G-MMI resins with different 2-BIB addition.

T. Liu et al. / Chemical Engineering Journal 304 (2016) 251–258

increased from 95 to 170 lmol/g gel when the amount of 2-BIB increased from 0.2 to 1.0 mL/g gel. G-MMI resins with different grafting densities were named as G95-MMI, G140-MMI, G170MMI, which corresponded to grafting densities of 95 ± 5, 138 ± 15, 170 ± 11 lmol/g, respectively. Secondly, GMA was grafted from the initiated agarose gel by surface-initiated AGET ATRP. Then MMI ligands were coupled by reacting with the epoxide group of poly(GMA) chains to prepare poly(GMA)-grafted HCIC resin G-MMI. The MMI ligand density increased from 365 to 400 lmol/g gel when the molar ratio of MMI to GMA increased from 1.0 to 3.0. Considering the high reaction activity of epoxide group with the mercapto group [34], the easier way to control the ligand density on G-MMI was to adjust the amount of epoxide group by controlling the GMA dosage during grafting. Thus, in the present work ligand density was proportional to the number of GMA units grafted in the resin, and the higher ligand density means longer grafting length for the same grafting density. Fig. 3 shows that the ligand density increased with the increase of GMA addition. For example, for the grafting density of 170 lmol/g gel, the ligand density increased from 165 to 1050 lmol/g gel when GMA addition increased from 0.05 to 0.60 mL/g gel. Moreover, the highest ligand coupling efficiency was achieved at high grafting density of 170 lmol/g. With the increase of grafting density, polymer chains were expected to form extended conformation [9,35], and the fully exposed active sites on the polymer chains would improve ligand coupling efficiency. The highest ligand density reached 1050 lmol/g for G170-MMI resins, which was higher than those of G140-MMI (780 lmol/g) and G95-MMI (580 lmol/g). The ligand density of G-MMI resins were also much higher than those of non-grafted HCIC resin MMI-L100 (100 lmol/g) and commercial HCIC resin MEP HyperCel (70–120 lmol/g). In this work, with surface-initiated AGET ATRP, grafting density can be adjusted by changing initiator precursor dosage (2-BIB), while ligand density can be controlled by varying the addition of monomer GMA. In addition, different kinds of functional ligands could be coupled onto the poly(GMA)-grafted chains by nucleophilic attack of ligands on epoxide groups [36]. Surface-initiated AGET ATRP as mentioned above could also be used for other bioseparation materials such as cellulose, polymeric and dextran matrices, membrane, monolith and so on, which certainly broaden the applications of the polymer-grafted materials for protein purification. Moreover, the functional ligands in the poly(GMA)grafted resins would distribute only on the grafting layer, which indicates less heterogeneity of ligand density than

Fig. 3. Ligand density of G-MMI resins with different GMA addition.

255

dextran-grafted resins. Most importantly, the polymer-grafted materials with suitable grafting density and ligand density could be designed accurately to improve the protein binding efficiency. The following evaluations, including static adsorption, adsorption kinetics and dynamic binding capacity, would reveal the importance of grafting control and verify the improvements of new resins prepared.

3.2. Protein adsorption equilibria Adsorption isotherms of hIgG on series of poly(GMA)-grafted GMMI and non-grafted MMI-L100 resins were measured and the results are shown in Figs. S1–S3 (Supporting information). It was found that the saturated adsorption capacities Qm were greatly relied on ligand density and grafting density (Fig. 4). For same grafting density resins, Qm values first increased and then decreased with the increase of ligand density. The highest Qm value could be found at the moderate ligand density of 300–500 lmol/g. When ligand density was low, the resins provided less accessible binding sites and relatively weak hydrophobic interactions for IgG adsorption. Meanwhile, ligand utilization might be low when ligand density was too high due to space limitation and interligand steric hindrance. It could also be found that there existed some cross-effects of ligand density and grafting density on the adsorption of hIgG onto G-MMI resins. As shown in Fig. 4, for higher grafting density resins, Qm value increased more significantly with the increasing ligand density in the rising zone of Qm. For example, for the grafting density of 170 lmol/g (G170-MMI), Qm increased about 100% with ligand density increasing from 165 to 365 lmol/g, which was higher than 17% for the grafting density of 95 lmol/g (G95-MMI) with ligand density increasing from 105 to 300 lmol/g. In addition, as mentioned above, the highest Qm value could be found at the optimized moderate ligand density for different grafting densities, which increased from 55 to 90 mg/g gel with the increase of grafting density from 95 to 170 lmol/g. With the increase of grafting density, the polymer chains might become more stretched [9], and thus the ligands distributed on the polymer chains would expose more feasibly in the pore, which improved the accessibility of binding sites for target protein. The results indicated that higher grafting density and proper ligand density would be favorable to improve protein adsorption. In general, by controlling the grafting density and ligand density, G-MMI resins had obviously higher Qm value

Fig. 4. Qm of hIgG on G-MMI resins with different grafting densities and ligand densities, and MMI-L100 resin.

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Fig. 5. K of hIgG on G-MMI resins with different grafting densities and ligand densities, and MMI-L100.

(90 mg/g) than that of MMI-L100 (52 mg/g) due to more accessible binding sites. The K values increased with the increase of ligand density for the three grafting densities tested when ligand density was lower than 500 lmol/g gel (Fig. 5). G-MMI resins had obviously higher K value (2.5 mL/mg) than that of MMI-L100 (0.4 mL/mg), which indicated stronger hydrophobic interactions between ligands on the grafted polymer chains and target protein.

protein diffusion. It was also found that there existed the crosseffects of ligand density and grafting density on the adsorption rate of hIgG onto G-MMI resin. With higher grafting density, De values increased more significantly with the increasing ligand density in the rising zones of De values. As shown in Fig. 6, for G170-MMI resins, De values increase about 3.84  1011 m2/s with ligand density increasing from 165 to 365 lmol/g, which was significantly higher than 0.86  1011 m2/s for G95-MMI resins with ligand density increasing from 105 to 300 lmol/g. The highest De values at the optimized ligand density increased sharply from 0.95  1011 to 4.41  1011 m2/s with the increase of grafting density from 95 to 170 lmol/g. The reason might be that the adjacent polymer chains become close enough, which lead to facilitated transport of adsorbed proteins by the swing of the flexible chains, named ‘‘chain delivery” effect [10,15]. However, the steric hindrance for protein diffusion became stronger with the excessive polymer chains in pores, especially at higher grafting density. Therefore, De values decreased more significantly with the increasing ligand density in the dropping zone of De values. For example, at the grafting density of 170 lmol/g, the De values decreased about 92.1% with the ligand density increasing 86.3% from 365 to 680 lmol/g, which was significantly higher than 47.4% at the grafting density of 95 lmol/g with the ligand density increasing 93.3% from 300 to 580 lmol/g. In general, by controlling ligand density and grafting density, De values for the optimized G-MMI resins were much higher (about 716.7%) than that of MMI-L100 (0.54  1011 m2/s) due to the flexibility of grafted polymer chains, higher accessibility of functional ligands and ‘‘chain delivery” effects.

3.3. Protein adsorption kinetics

3.4. Dynamic binding capacity

The hIgG adsorption kinetics curves with G-MMI resins are shown in Figs. S4–S6 (Supporting information). Fig. 6 indicates that the calculated effective pore diffusivity De was greatly dependent on grafting density and ligand density. For resins with same grafting density, De values increased firstly and then decreased with the increase of ligand density. When ligand density was low, the polymer-grafted resins provide less ligand accessibility for mass transport of protein, and weaker hydrophobic interactions would cause stronger surface binding resistance for pore diffusion. However, when ligand density was too high, the grafted polymer chains would block pore volume, and caused strong steric effect for

The column breakthrough curves of hIgG with the optimized G-MMI resins (G95-MMI-L300, G140-MMI-L445 and G170-MMIL365), MMI-L100 and MEP HyperCel at varying linear velocities (100–300 cm/h) are showed in Figs. S7–S11 (Supporting information). DBC values at 10% breakthrough (Q10%) are listed in Table 1. The results show that at linear velocity of 100 cm/h DBC values increased obviously from 13.4 to 37.8 mg/g with the grafting density rising from 95 to 140 lmol/g, and kept stable at 37.4 mg/g for G170-MMI-L365. With the increase of linear velocity from 100 to 300 cm/h, the values of DBC decreased slightly for three G-MMI resins tested, and DBC value decreased only by 8.5% for G140-MMI-L445, Table 1 Comparison of the Q10%, Q10%/Q⁄ and Q10%/Qm of hIgG onto G95-MMI-L300, G140MMI-L445, G170-MMI-L365, MMI-L100 and MEP HyperCel resins at different linear velocities.

Fig. 6. De of hIgG on G-MMI resins with different grafting densities and ligand densities, and MMI-L100.

HCIC resin

U (cm/h)

Q10% (mg/g gel)

Q10%/Q⁄ (%)

Q10%/Qm (%)

G95-MMI-L300

100 200 300

13.4 12.8 12.2

37.3 35.6 34.0

22.5 21.6 20.6

G140-MMI-L445

100 200 300

37.8 36.0 34.6

64.0 62.3 59.8

58.2 55.4 53.1

G170-MMI-L365

100 200 300

37.4 31.0 27.5

52.8 43.6 39.0

41.5 34.3 30.5

MMI-L100

100 200 300

4.7 4.4 3.6

20.3 19.1 15.3

9.1 8.5 6.7

MEP HyperCel

100 200 300

38.8 22.9 15.5

36.2 21.3 14.4

34.5 20.3 13.7

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which might be attributed to its strong hydrophobic interactions provided by the highest ligand density (445 lmol/g) and suitable grafting density (140 lmol/g). In general, G140-MMI-L445 showed the highest DBC value (34.6 mg/g) at linear velocity of 300 cm/h, which was 861%, 123% and 86% higher than nongrafted resins MMI-L100, MEP HyperCel and dextran-grafted HCIC resin MMI-B-XL-200 [10]. Furthermore, the ratios of Q10% to Q⁄ at loading hIgG concentration of 2.0 mg/mL (Q10%/Q⁄) and the ratios of Q10% to Qm (Q10%/Qm) were used to evaluate the dynamic adsorption efficiency in the chromatographic separation. As listed in Table 1, Q10%/Q⁄ and Q10%/Qm of G140-MMI-L445 were 64.0% and 58.2% at the linear velocity of 100 cm/h, which were significantly higher than 20.3% and 9.1% for MMI-100, and 36.2% and 34.5% for MEP HyperCel, respectively. The results indicate that G-MMI resins with the optimized grafting density and ligand density could provide strong binding force and rapid mass transport for protein adsorption. Although the affinity between G-MMI resins and IgG was higher significantly than other non-grafted MMI resins, it was substantially lower than Protein A and ion-exchange resins. For example, the optimized K values in this work were 1.7–2.5 mL/mg, but the K value on Protein A for IgG was 71.4 mL/mg [37], and the K value of SP-H (ion-exchange resin) for IgG was 76.9 mL/mg [13]. The suitable value of K would be beneficial for the protein elution at relative mild conditions. The separation performance of G-MMI resin and protein recovery would be evaluated further with protein mixture. 3.5. hIgG separation from protein mixture The resin G170-MMI-L365 was used to separate hIgG from a protein mixture (1 mg/mL hIgG and 4 mg/mL HSA) to evaluate the selectivity of new G-MMI resin in a more complex system. The protein mixture was loaded at pH 7.0 and the effect of elution pH (4.0, 4.5, 5.0 and 5.5) on the purity and recovery of hIgG was investigated. The chromatograms are shown in Fig. S12 (Supporting information), and the SEC-HPLC analysis of loading, breakthrough and elution is shown in Figs. S13–S14 (Supporting information). It could be found that almost all HSA flowed through the column and no hIgG was detected in the breakthrough fractions, which indicated high selectivity and adsorption ability of new G-MMI resins for hIgG. Low recovery of hIgG was found at the elution pH 5.5. The pKa of MMI ligand is 5.3 [15], so the electrostatic repulsion between hIgG and MMI ligand at pH 5.5 would be too weak to desorb hIgG, resulting in very low recovery of hIgG. When pH was at the range of 4.0–5.0, the electrostatic repulsion between the protonated imidazole groups of MMI ligand and the positive charged proteins would become strong and overcame the hydrophobic attractions, resulting in better elution and high recovery. Best recovery (96.9%) and high purity (99.8%) of hIgG were found at elution pH 5.0, which were better than those of MEP HyperCel and W-ABI resin with the similar feedstocks [32]. The elution at pH 5.0 for G-MMI resin was significantly milder than the elution at pH 3.0–3.5 for Protein A-base resins due to the relative weak affinity between G-MMI resin and hIgG as mentioned above, which is beneficial to protect antibody activity and reduce antibody aggregation under acidic conditions [38,39]. 3.6. Reusability of G-MMI resin The reusability of G170-MMI-L365 resin was tested with same procedure of hIgG separation from protein mixture (1 mg/mL hIgG and 4 mg/mL HSA) with 50 cycles. The protein mixture was loaded at pH 7.0 and hIgG was eluted at pH 5.0. The purity and recovery of hIgG for each cycle are shown in Fig. 7. During the 50 cycles, hIgG purity was quite stable and kept at the range of 98.5–99.8% with

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Fig. 7. Recovery and purity of hIgG with G170-MMI-L365 separation from protein mixture (hIgG/HSA = 1:4) for 50 cycles. Separation conditions: loading at pH 7.0 and elution at pH 5.0.

the average of 99.3%. The recovery was also as high as 90.2– 96.9% with the average of 92.9%. In addition, after 30 and 50 cycles the DBC values for hIgG were tested at linear velocity 100 cm/h. It was found that Q10% values were quite stable and kept 97.1% after 30 cycles and 95.3% after 50 cycles. The results demonstrated that new G-MMI resins would be suitable for the industrial applications. 3.7. Removal of HCP and DNA from cell culture supernatant The G170-MMI-L365 resin was further challenged to separate mAb from CHO cell culture supernatant, and the removals of HCP and DNA were determined. The log reduction values (LRV) of HCP and DNA for G170-MMI-L365 resin were 0.8 and 3.2, respectively. The LRV about HCP removal for G170-MMI-L365 resin developed in the present work was higher than 0.6 of MEP HyperCel resin, and was similar to 0.76 of multimodal charge-induction chromatographic resin W-ABI [32]. 4. Conclusions Surface-initiated AGET ATRP was explored as a controlled polymerization method to prepare new polymer-grafted chromatographic resins. Grafting density and ligand density of poly(GMA)grafted HCIC resin G-MMI could be controlled by adjusting the amounts of activator 2-BIB and monomer GMA, respectively. The cross-effect of ligand density and grafting density on the hIgG adsorption to G-MMI resins was studied. Higher grafting density and proper ligand density are critical to ensure the formation of three-dimensional binding scaffold that results in high protein adsorption capacity. Protein breakthrough experiments indicated that G-MMI resins with the optimized grafting and ligand densities could be used for high linear velocity. DBC value at the linear velocity of 300 cm/h was 861.1% and 86.3% higher than non-grafted resin MMI-L100 and dextran-grafted resin MMI-B-XL-200, respectively. The selectivity and reusability of new G-MMI resins were tested with the separation of hIgG from a model protein mixture containing HSA. High purity and recovery of hIgG were obtained for 50-cycle separation, and the mild acidic condition (pH 4.0– 5.0) would be suitable for the effective elution of hIgG. In general, surface-initiated AGET ATRP leaded to the controlled grafting and achieved high-capacity adsorption and high-effective separation

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with new HCIC resins, which had a potential application for largescale antibody purification. Future works would be focused on the design of series of polymer-grafted resins with different pore sizes and functional ligands by surface-initiated AGET ATRP and test the crude resources (such as serum and mammalian cell culture broth) to expand its application for protein separation.

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

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Appendix A. Supplementary data [22]

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cej.2016.06.074.

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