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Fabrication of poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) macroporous microspheres through activators regenerated by electron transfer atom transfer radical polymerization for rapid separation of proteins
Journal of Chromatography B 1128 (2019) 121794
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Fabrication of poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) macroporous microspheres through activators regenerated by electron transfer atom transfer radical polymerization for rapid separation of proteins
T
Ning Daib,1, Shaoyun Wangd,1, Heng Lia, Lan Zhaoc, Haibo Jina, Ning Ana, Pisheng Gonga, ⁎ Qiqi Tana, Xu Tanga, Fei Wanga, Rongyue Zhanga, a
Beijing Key Laboratory of Fuels Cleaning and Advanced Catalytic Emission Reduction Technology/College of Chemical Engineering, Beijing Institute of Petrochemical Technology, Beijing 102617, China Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China c National Key Lab of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China d Senhui Microsphere Tech (Suzhou) Co., Ltd., Suzhou 215123, China b
ARTICLE INFO
ABSTRACT
Keywords: Macroporous microspheres Suspension polymerization Atom transfer radical polymerization Separation of proteins
Activators regenerated by electron transfer atom transfer radical polymerization (AGET ATRP) were firstly used in suspension polymerization to prepare macroporous microspheres based on a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate. Compared to conventional radical polymerization (CRP), the microspheres by AGET ATRP showed more homogeneous structure, larger pores, and higher protein binding capacity. The body of microspheres are formed by the large clusters resulted from the aggregated little particles. The size of the particles in microspheres by AGET ATRP was 10–300 nm which was smaller than that (400–800 nm) of the microspheres by CRP. AGET ATRP gave larger pore size (275 ± 5 nm) and surface area (59.3 ± 1 m2/g) than CRP (234 ± 5 nm, 37.5 ± 1 m2/g). The microspheres were modified with polyethylene imine for anion resins that were evaluated in term of its protein binding capacity. The results indicated that the static (69 ± 0.5 mg/mL) and dynamic binding capacity (61 ± 0.5 mg/mL) of proteins on modified microspheres by AGET ATRP were higher than that (34 ± 0.5 mg/mL and 19 ± 0.5 mg/mL) by CRP. Meanwhile, the proteins binding capacity on the microspheres by AGET ATRP decreased only less than 10% when the flow rate increased 10 times. These macroporous media show a large potential in rapid separation of proteins.
1. Introduction Polymer microspheres, as an attractive chromatographic medium, have been widely used for separation and purification of proteins due to its good mechanical strength and chemical stability. Generally, the large pores of the media play a major role in realizing high throughput chromatography. It is well known that the large pore size is advantageous in separation for rapid mass transport [1,2]. However, the larger pores usually cause the smaller surface areas in the porous microspheres. The surface areas of the macroporous microspheres will decrease to less than 60 m2/g when its average pore size is more than 100 nm, and even be reduced to less than 10 m2/g [3]. As a result, low protein binding capacity is often observed on the chromatographic
media with large pores [4]. Therefore, it has been an attractive topic for researchers to improve the protein binding capacity on the chromatography with large pores. As a common method, grafting polymers in these media was usually used for increasing the capacity. In our previous study [5], coating branched polyethyleneimines (PEI) on the gigaporous microspheres (based on a copolymer of glycidyl methacrylate and divinyl benzene, PGMA-DVB) was conducted and the protein binding capacity had a rising trend with the amount of PEI in the range of 0.11–0.24 mmol/mL ion exchange capacity. Meanwhile, it was found that the capacity began to decrease when the ion exchange capacity was more than 0.24 mmol/ mL. The possible reason was that extra PEI chains reduced surface area of microspheres, so, the adsorption sites on the PEI were buried and
Corresponding author. E-mail address: [email protected] (R. Zhang). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.jchromb.2019.121794 Received 5 June 2019; Received in revised form 11 August 2019; Accepted 7 September 2019 Available online 10 September 2019 1570-0232/ © 2019 Elsevier B.V. All rights reserved.
Journal of Chromatography B 1128 (2019) 121794
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could not bind proteins. In reported methods, it also was found that the protein binding capacity reached a constant value or began to decrease when the amount of grafting polymers exceeded a certain value [6,7]. As an alternative way, Svec's group [8] used nanoparticles to coat the polymer monolith for increasing its surface area, and a high column efficiency was obtained in this modified monolith while the reducing pore size increased column pressures. The above-mentioned methods could be summarized as the same approach, in which the protein binding capacity could be improved through modifying the surface of the macroporous matrix with multifunctional compounds to increase adsorption sites. In this way, the structure of the media did not change except for slightly reducing the pore sizes due to the modifying compounds occupying some volume in the media. However, capacity was limitedly promoted owing to the reduced surface area of the matrix. Therefore, fabrication of macroporous supports with high surface area should be an alternative method for increasing protein capacity without sacrificing large pores. The macroporous microspheres are generally prepared though suspension polymerization initiated by conventional radical polymerization (CRP) [9]. In this method, polymerization takes place in each emulsion drop and the formation of pores depends on the process of phase separation between polymers and porogens. Therefore, screening reaction conditions and porogens are always concerned by many researchers [10,11] for large pores, but the matrix structure of the microspheres is not remarkably changed due to the nature of CRP. In this way, the growing polymer chains in CRP tend to aggregate each other because van der Waals attraction surmounts the steric hindrance mutually expelling the polymer chains [12], and the segregated polymer chains soon form tiny particles, then those particles aggregate to the typical heterogeneous macroporous structures composed of micron size global particles [13,14]. The micron size particles and random aggregation led to low surface area and heterogeneous structure of the supports. To address this problem, we ever used atom transfer radical polymerization (ATRP) to fabricate the monolith and found that ATRP could get higher surface area and more homogenous structure [15] than CRP. It is also proved by polymer gels prepared through ATRP that are more homogeneous than ones by CRP [16]. In comparison with CRP, the concentration of propagating radicals is low during polymerization in ATRP [17]. Hence, it often takes hours for an individual chain to propagate hundreds of monomer units, and thus chains growth through propagation is slowed down [18]. In contrast to this, only seconds for the same number of monomer units are needed to be added to the polymer chain in CRP while the process of chain relaxation would need minutes or hours. Therefore, the larger micro particles would be formed in CRP than those in ATRP and resulted in low surface area and heterogeneous structure. Based on the above description, the macroporous microspheres are expected to afford a high surface area and protein binding capacity through suspension polymerization initiated by ATRP. The aim of this study is to explore suspension ATRP and fabricate macroporous microspheres with great surface area, large pores, and high protein binding capacity.
Dichloromethane (DCM), n-octanol (OA), poly(vinyl alcohol) (PVA, Mw = 1700 ± 50, 88% alcoholysis degree), sodium lauryl sulfate (SLS), ascorbic acid (AC) and CuBr2 were purchased from Beijing Chemical Plant (Beijing, China). Poly(ethylene imine) (PEI, Mw = 600) was obtained from XIYA company (Shandong, China). Lysozyme, ovalbumin, proteins marker, and bovine serum albumin (BSA) was purchased from Sigma–Aldrich (St. Louis, MO, USA). All the above chemicals were received. Other reagents used in experiments were of analytical grade and bought from Beijing Chemical Factory (Beijing, China). 2.2. Preparation of PGMA-EDMA microspheres by AGET ATRP In a typical polymerization, GMA (5.0 mL, 38.5 mmol), EDMA (5.0 mL, 26.5 mmol), EBP (0.017 mL, 1.3 mmol), CuBr2 (0.29 g, 1.3 mmol), DCM (15.0 mL) and OA (15.0 mL) were added to an ampule. The ampule was then sealed with a rubber septum and degassed with nitrogen for 20 min. Deoxidized PMDETA (0.27 mL, 1.3 mmol) was quickly added to the ampule by a degassed syringe. The ampule was shaken for 30 s and the reaction mixture was subsequently injected into the reactor containing 160 mL aqueous solution composed by 4.8 g PVA, 1.5 g ascorbic acid and 0.16 g SLS under 210 rpm stirring. The reactor was heated to be 60 °C and the polymerization proceeded at 60 °C for 8 h. The reaction solution was filtered and successively washed with deionized water and ethanol; the resulted microspheres were then extracted in a Soxhlet apparatus with acetone and deionized water for 48 h, respectively. Finally, the microspheres were stored in 20% ethanol aqueous solution at room temperature. As a parallel experiment, the same processes were performed by conventional radical polymerization initiated by AIBN. In these comparative experiments, all of reaction conditions and the following treatments were as the same as AGET ATRP except for no CuBr2 added in these reaction systems. 2.3. Modification of PGMA-EDMA microspheres with PEI
2. Material and methods
In order to measure the protein binding capacity on the PGMAEDMA microspheres, PEI was used to modify the supports for anion exchange media. This derivation was carried out according to the previous study [5]. The typical procedure was shown in Scheme 1 and began with copolymerization of GMA and EDMA by AGET ATRP at 60 °C for 8 h, followed by attaching PEI with required molecules to PGMA-EDMA microspheres in dimethyl sulfoxide (DMSO) for 24 h at 60 °C. The ionic capacity of modified microspheres (PGMA-EDMA-PEI) was determined by chloride-silver nitrate titration as described by Sun et al. [19]. The modified microspheres (10 mL) were filled into a glass column, and then was washed with 10 column volumes (CVs) of 1.0 mol/L NaCl to saturate the ion exchange sites with chloride ions and rinsed with 0.1 mmol/L HCl. Then, the column was rinsed with five CVs of Na2SO4 (10%, w/w), during which the bound chloride ions were replaced with sulfates. Chloride ions in the effluent were titrated with 50 mmol/L AgNO3 in the presence of K2CrO4 (0.5 mL, 5%, w/w) as an indicator. Then, the IC of the column was calculated from the titration data.
2.1. Materials
2.4. Pores distribution and morphology of the microspheres in dry state
Glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) were purchased from Sigma–Aldrich (St. Louis, MO, USA) and purified before use. Ten dextran standards of molecular weights ranging from 342 to 2,285,000 Da were obtained from Sigma–Aldrich (St. Louis, MO, USA). Azobisisobutyronitrile (AIBN) was produced by Shanghai Chemical Plant (Shanghai, China) and refined by recrystallization from the methanol before use. N,N,N′,N″,N″-pentamethyl diethylenetriamine (PMDETA) was purchased from JK Chemical Ltd. (Beijing, China). Ethyl-2-bromopropionate (EBP) was obtained from Alfa Aesar (Lancs, England). DEAE-Sepharose fast flow was from GE company (USA).
Pore size distribution of PGMA-EDMA by two methods, including AGET ATRP and CRP, as well as PGMA-EDMA-PEI, in their dry state, were determined by an Auto Pore IV 9500 mercury porosimeter (Micromeritics, USA). The specific surface area and porosity of the samples were measured by N2 adsorption on a Micromeritics ASAP2020 analyzer (Micromeritics, USA) and calculated by the Brunauer-EmmettTeller (BET) method. The morphologies of these microspheres were observed by scanning electron microscope (SEM) (JSM-6400, JEOL Lim. Co., Japan). The detailed method of sample preparations for SEM observation was described as following. Microspheres were re2
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Scheme 1. Preparation and modification of PGMA-EDMA microspheres.
suspended in distilled water. Then, the dispersion was dropped on a piece of aluminum foil and dried at an ambient atmosphere. The sample was placed on a metal stub with double-sided conductive adhesive tape, and then it was coated with a thin gold film under reduced pressure below 5 Pa with a JFC-1600 fine coater (JEOL, Japan).
2.6. Determination of static and dynamic binding capacities Static binding capacity (SBC) experiments were carried out in the equilibration buffer (20 mmol/L Tris-HCl buffer, pH 8.0) using the method in the previous report [5]. The typical procedure was performed as the following: 0.1 g microspheres was firstly equilibrated with the buffer and then was mixed with 10 mL of protein solution (BSA, 0.3–3.0 mg/mL) prepared in the same buffer and incubated at 120 rpm and 21 °C for 24 h. The supernatant was subsequently collected by centrifuging at 5000 rpm for 5 min and protein concentration (c) in the supernatant was finally measured at 280 nm using a UV transparent 384 well plate (Brand, Wertheim, Germany) and the adsorption capacity of protein (qS) was calculated by mass balance,
2.5. Measurement of pore distribution of the microspheres in wet state by inversed size exclusive chromatography (ISEC) The microspheres were packed into the columns (ø10 × 500 mm) and the final packed bed height was 50 cm. The parameters of the dextran, such as the weight-average molecular weight Mw, the numberaverage molecular weight Mn, the molecular weight corresponding to the elution peak Mp, the viscosity radius Rη, and the polydispersity (Mw/Mn), were shown in Table 1 [20]. Dextran solutions were prepared by dissolution in the operating buffer (200 mM NaCl, 20 mM Tris-HCl solution, at pH 7.0) and filtered through 0.22 μm membrane to remove possible aggregates prior to injection into the columns. Waters 2695 equipped with 2414 refractive index and 2696 photodiode array detector (Waters, Milford, MA, USA) was used to determined chromatography behaviors of the microspheres at room temperature. ISEC was performed at 1.0 mL/min, using injection volumes of 100 μL. The interparticle volume V0 was determined using monodisperse nano-SiO2 particles (80 nm) as the excluded solute. The total mobile phase volume VT was measured using glucose. The SEC distribution coefficient, Kd, was measured by the following equation:
Kd =
Ve
V0 Vp
=
Ve VT
V0 V0
qS =
qD =
Mna
Mw/Mna
Rη (nm)b
1500 5000 12,000 25,000 50,000 80,000 150,000 270,000 410,000 670,000 3,800,000
1080 4440 9890 21,400 43,500 66,700 123,600 196,300 276,500 401,300 2,285,000
1350 5220 11,600 23,800 48,600 80,900 147,600 273,000 409,800 667,800 3,790,000
1160 3260 8110 18,300 35,600 55,500 100,300 164,200 236,300 332,800 1,500,000
1.16 1.6 1.43 1.30 1.36 1.46 1.47 1.66 1.73 2.01 2.53
0.88 1.78 2.65 3.89 5.53 6.845 9.31 11.72 13.90 16.73 39.78
a b
V0 ) × c V
(3)
2.7. Transport of proteins in the microspheres by CLSM The transport of labeled proteins in the microspheres was monitored by TCS SP2 confocal laser scanning microscope (CLSM) (Leica, Germany). Fluorescein isothiocyanate-labeling hepatitis B surface antigen (FITC-HBsAg) adsorption experiments were conducted in a 4 °C incubator, after 12 h adsorption, the microspheres were separated from the FITC-HBsAg solution by centrifugation and washed with 50 mM Tris-HCl buffer three times to remove desorbed protein molecules completely. After being placed on a slide glass and being covered with cover glass, the microspheres were observed with LCSM to visualize the adsorption of FITC-HBsAg onto microspheres. The samples were detected at 488 nm, and the fluorescent images at 520–550 nm wavelengths were then taken.
Table 1 Molecular weights (Mp, Mw, Mn), polydispersity (Mw/Mn) and viscosity radii (Rη) of the dextran standards used in ISEC. Mwa
(V1
where V1 is the 5% breakthrough volume, V0 is the sum of void volumes of column and HPLC system, c is the concentration of protein (mg/mL), and V is the volume of media in column (mL).
where Ve was the elution volume of the analyte and Vp was interparticle volume.
Mpa
(2)
where qm is the maximum adsorption capacity and K is the Langmuir dissociation constant. The isothermal data was fitted using Sigma Plot 8 (Systat Software Inc., San Jose, CA, USA). Dynamic binding capacity (DBC) was determined by frontal analysis experiments according to the reported method [5]. Frontal analysis experiments were performed at pH 8.0 with 1.0 mg/mL BSA in pH 8.0 Tris-HCl buffer. The dynamic binding capacity (qD) was calculated by Eq. (3)
(1)
Dextran standard
qm × c K+c
2.8. Separation of proteins Separation of model proteins mixture (including lysozyme, ovalbumin, bovine serum albumin) processes were carried out using 20 mM
All of data was from quality inspection of productions. Rη were calculated using the correlation Rη = 0.0271×Mp0.498 [20]. 3
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3.2. Effect of polymerization temperature on morphology of microspheres
Tris-HCl pH 8.0 as the running buffer A and 20 mM Tris-HCl 1 M NaCl pH 8.0 as the elution buffer B. PGMA-EDMA-PEI was packed into a column (Ф10 × 300 mm) with a final bed height of 25.4 cm and a bed volume of 20 mL. Before loading, the column was equilibrated for 5 column volume (CV) with buffer A. After loading, the column was washed with buffer A for 2.5 CV and then eluted by a linear gradient elution to 100% buffer B with a gradient length of 12 CV at the flow rate of 5 mL/min. Fractions of the flow-through as well as the eluates were collected and further analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyacrylamide-based gel was prepared according to manufacturer's instructions using Liu Yi DYCZ-28B electrophoresis (Beijing Liu Yi, Beijing, China). After completion of running the gel at 100 V, the gel was removed from the tank and stained with Coomassie dye (Thermo Fisher Scientific, Waltham, MA, USA). The gel was washed with DDI water until all protein bands were clearly observed. SDS-PAGE images were obtained by camera.
Polymerization temperature is often used for controlling the pore size distribution in polymer porous materials. In convention radical polymerization (CRP), the lower the polymerization temperature, the larger the pores [3]. Keeping other condition fixed (1:1 GMA/EDMA ratio, 3:1 porogen/monomers ratio, 1:1 DCM/OA ratio), the temperature varied from 55 to 70 °C. Because the boiling point of DCM is 39.8 °C and will be lost in high temperature, more than 70 °C is not be considered in this experiment. Fig. 2 shows the effect of temperature on the morphology of microspheres prepared at 55 °C (A1, C1), 60 °C (A2, C2), 65 °C (A3,C3) and 70 °C (A4, C4). In comparison to CRP (Fig. 2 C14), AGET ATRP (Fig. 2 A1-4) showed a similar trend of decrease of pores size with temperature, but no significant changes of morphology were observed on the microspheres by AGET ATRP, and the interconnected network structure was still retained. On the contrast, CRP showed that the size of aggregation particles had a great decrease with temperature (Fig. 2C1-4). Meanwhile, no interconnected network in microspheres by CRP can be found at each temperature. As indicated above, it was found the pore size and pores interconnectivity decreased with reaction temperature. Generally, the large pore and high interconnectivity were the key factors for realizing fast transport of proteins. The formation of microspheres that consumed less than 10 min was faster than CRP (4 h) at 60 °C. In order to obtain high throughput matrix, 60 °C is considered for the optimal polymerization temperature.
3. Results and discussion 3.1. Effect of GMA to EDMA ratio on morphology of microspheres GMA to EDMA ratio had an influence on the morphology of microspheres [21]. The polymerization was conducted in the range from 2:1 to 0.5:1 GMA/EDMA ratio, and the others conditions were constant in all polymerization, including porogen to monomers ratio (3:1, v/v), porogenic solvent (1:1 (v/v) dichloromethane (DCM) /n-octanol (OA) ratio), temperature (60 °C) and reaction time (8 h). In order to compare CRP and AGET ATRP, the same polymerizations conditions in AGET ATRP were carried out as those in CRP with AIBN as initiator. As shown in Fig. 1, Fig. 1 A1-A4 were the morphology of microspheres from AGET ATRP and the size of aggregation particles was reduced with the decrease of GMA/EDMA ratio. Meanwhile, the network of microspheres became more homogenous. In microspheres by CRP, the similar trend was also observed (Fig. 1 C1-C4), but the heterogenous structure was higher than microspheres by AGET ATRP at each reaction condition. Although, the low GMA/EDMA ratio can afford homogeneous network, and a low GMA would decrease the content of reactive epoxide groups that are needed for the subsequent functionalization of the microspheres. To obtain microspheres with homogenous structure and enough reactive epoxide groups, the 1:1 GMA/EDMA ratio is selected for the optimal ratio.
3.3. The effect of porogen to monomers ratio on the morphology All of both type of microsphere from AGET ATRP (A1-3) and CRP (C1-3) afford increased pores size and pore volume with the porogen/ monomer ratio. Generally, the pores of the microspheres are from the voids within the porous beads and the fraction of voids is close to the volume fraction of the porogen in the initial polymerization mixture. Compared to the effect of GMA/EDMA and temperature, the porogen/ monomer ratio had significant influence on microspheres by AGET ATRP [22–24]. As might be expected, microspheres by AGET ATRP (Fig. 3A1-3) gave more pores and better interconnected pores in comparison to CRP (Fig. 3. These features of microspheres by AGET ATRP are advantageous in promoting rapid mass transfer through a porous microsphere. Meanwhile, the large pore size and high surface area can be expected in these porous microspheres. The polymerization system
Fig. 1. SEM of microspheres at different GMA/EDMA ratio, A1-4:2:1, 1.5:1, 1:1, 0.5:1, by AGET ATRP; C1-4: 4:2:1, 1.5:1, 1:1, 0.5:1, by CRP. 4
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Fig. 2. SEM of microspheres at various temperatures, A1-4: 55, 60, 65, 70 °C, by AGET ATRP; C1-4, 55, 60, 65, 70 °C, by CRP.
was designed according to the conditions, including 1:1 GMA/EDMA, 3:1 porogen/monomers, 1:1 DCM/OA ratio, 60 °C and 8 h. All of microspheres in the following study, as the matrix, is prepared by this polymerization condition if no other description is cited.
Table 2 The structure parameters of microspheres by mercury intrusion.
3.4. Comparison of microspheres by AGET ATRP and CRP
Supporta
Total intrusion volumeb (mL/g)
Average pore diameter (nm)b
Surface area (m2/g)c
Porosity (%)b
AGET ATRP CRP
3.71 ± 0.5 1.78 ± 0.5
275 ± 5 234 ± 5
59.3 ± 1 37.5 ± 1
88.5 ± 2 69.8 ± 2
a All of microspheres was prepared by the same reaction conditions: 1:1 GMA/EDMA, 3:1 porogen/monomers, 1:1 DCM/OA ratio, 60 °C and 8 h. b These data were measured by mercury intrusion method. c These data were determined by N2 adsorption method.
Based on the optimized polymerization conditions, the microsphere structures, including pore size, surface area, porosity, and morphology, were compared between AGET ATRP and CRP. The relative results were shown in Table 2 and Fig. 4, respectively. Compared to CRP, all of structure parameters of microspheres by AGET ATRP, including average
Fig. 3. SEM of microspheres at various porogen/monomer ratio, A1-3: 1:1, 2:1, 3:1, by AGET ATRP; C1-3, 1:1, 2:1, 3:1, by CRP. 5
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Fig. 4. SEM of microspheres by AGET ATRP (1) and CRP (2).
Scheme 2. Gel formation in the droplets by two ways of AGET ATRP and CRP.
Fig. 5. The backpressures on PGMA-EDMA at various flow rates.
Fig. 6. Pore distributions of microspheres by mercury intrusion.
pore diameter, surface area, and porosity, was higher. Especially, both the average pore and surface area by AGET ATRP (275 ± 5 nm, 59.3 ± 1 m2/g) were higher than those from CRP (234 ± 5 nm, 37.5 ± 1 m2/g). The usual rule was well known that the larger pore size usually caused the lower surface area [25]. However, it was found that the surface area of the microspheres by AGET ATRP (275 nm pore size) was more 58% than CRP with 234 nm pore size. This abnormal result aroused us interest in exploring the structure difference between these two microspheres by AGET ATRP and CRP. The morphologies and of PGMA-EDMA by AGET ATRP and CRP
were observed through scanning electronic microscopy (SEM) and shown in Fig. 4. In Fig. 4, Fig. 4-1 (AGET ATRP) showed that the polymer skeleton was composed by hundred-nanometer particles (10–300 nm) that was smaller than CRP (400–800 nm, Fig. 4-2). Meanwhile, higher porosity and inter-connectivity could be observed on the Fig. 4-1 than those from PGMA-EDMA by CRP (Fig. 4-2). Generally, the small particles or clusters bring large surface area [25]. PGMA-EDMA by AGET ATRP therefore showed higher surface area than CRP. According to the general rule that larger particles or clusters give larger pores, microspheres by CRP could have afforded larger pores 6
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Table 3 Static and dynamic binding capacity of PGMA-EDMA-PEI microspheres. Support
QS (mg/mL)
a
QD (mg/mL)
b
QD (mg/mL)
c
QD (mg/mL)
d
PGMA-EDMA-PEI (AGET ATRP) PGMA-EDMA-PEI (CRP)
69 ± 0.5
61 ± 0.5
59 ± 0.5
58 ± 0.5
56 ± 0.5
34 ± 0.5
19 ± 0.5
14 ± 0.5
13 ± 0.5
11 ± 0.5
QD (mg/mL)
a,b,c,d
The dynamic binding capacities were determined at the flow rate of 361 cm/h, 1444 cm/h, 2527 cm/h, and 3610 cm/h, respectively.
than AGET ATRP. On the contrary, microspheres by AGET ATRP showed larger pores than CRP. The possible reason was that the high interconnectivity contributed to large pore and porosity in microspheres by AGET ATRP. These differences should be relative with way of polymerization. In our previous preparation of monolith by AGET ATRP, the obtained monolith showed three-dimension bicontinuous skeleton while the monolith by CRP was composed from aggregation of particles under the same conditions [15] as ATRP. Kanamori also reported the similar result and the monolith by living polymerization could obtain three dimensional bicontinuous skeleton structure [26]. The similar explanation for formation of this structure was that living polymerization gave more homogeneous gels than CRP. In present study, ATRP suspension polymerization was used to prepare microspheres, which differed from in-situ polymerization in effect of the tension at liquid-liquid interface [3] not in gel structure formation. In suspension polymerization, the gel is formed in each droplet that is considered as a reaction unit. The droplet, including reactive composition, porogen and initiator, formed the microsphere after polymerization. Meanwhile, each microsphere is also considered as a gel that is formed from cross linked microgels (tiny particles). The resulted structure of microsphere mainly depended on the microgel size and how to be crosslinked. The formations of gels by the two ways, ATRP and CRP, were described by Scheme 2. As shown in Scheme 2, the gel formation began with polymerization in a droplet, then many polymer chains with similar molecular weights were formed by ATRP and dispersed in the porogen solvent prior to crosslinking reaction (or in the early stage of polymerization), which was attributed to the nature of living/control polymerization (well controlling molecular weight of polymer); these polymer chains were followed by crosslinking reaction with further polymerization and homogenous gel was finally formed. In CRP, many micro gels with different sizes were developed in the early polymerization stage due to the nature of CRP [27,28]; the micro gels aggregated and formed larger gel particles (clusters) due to local fluctuation of micro gels concentration with polymerization. These
Fig. 8. Pore distributions of microspheres by ISEC.
particles (clusters) were crosslinked and resulted in the heterogeneity of the resulted gels. This heterogeneity led to low connectivity between the pores in the microspheres. 3.5. Permeability of PGMA-EDMA microspheres In the case of porous materials, high porosity and connectivity mean good permeability or high throughput for separation. To prove this point, the permeability of PGMA-EDMA was determined by evaluating their backpressures under different flow rates. PGMA-EDMA microspheres were filled in the stainless columns (ø10 × 50 mm). Fig. 5 showed that the backpressures of two columns had a linear increase with flow rates (in the range of 361–2889 cm/h), and this indicated that good mechanical strength could be kept under the testing pressures [29]. Meanwhile, PGMA-EDMA by AGET ATRP had lower back pressure than ones by CRP at each flow rate. This proved that PGMA-EDMA by AGET ATRP had better permeability than that by CRP. These comparisons further verified the observation from SEM, the pores in PGMAEDMA by AGET ATRP had better interconnectivity than those by CRP. 3.6. Modification of PGMA-EDMA microspheres by PEI From the above-mentioned results, it could be found that AGET ATRP could give higher surface area and permeability than CRP. Next, it was explored whether these microspheres by AGET ATRP could give
Fig. 7. Dynamic binding capacity on PGMA-EDMA-PEI at different flow rates, (A, microspheres by AGET ATRP, B, microspheres by CRP). 7
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Fig. 9. LSCM of HBsAg on PGMA-EDMA-PEI by AGET ATRP and CRP, A1 and A2, AGET ATRP; B1 and B2, CRP.
Fig. 10. Separation of proteins on PGMA-EDMA-PEI and SDS-PAGE of the corresponding chromatographic peak fractions. Peak a, lysozyme, b, ovalbumin, c, BSA; Lane a, b, c: the collected fraction of peak a, b, and c, respectively; Lane M, proteins marker.
higher protein binding capacity. To examine this, the microspheres were modified by PEI to obtain anion exchange supports. PEI was used for modification of gigaporous microspheres (poly(glycidyl methacrylate-co-divinyl benzene)) to separation of proteins in our previous study [5]. The ion capacities of modified PGMA-EDMA microspheres were
determined and the results were 0.21 mmol/mL (PGMA-EDMA by AGET ATRP) and 0.23 mmol/mL (PGMA-EDMA by CRP). The effect of modification on the microspheres structure was also an important item to be concerned in our study. The pore distribution of modified microspheres was determined by mercury intrusion method and shown in 8
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Fig. 6. The average pore and pore volume of PGMA-EDMA-PEI and PGMA-EDMA by AGET ATRP were 268 ± 5 nm, 275 ± 5 nm, 3.50 ± 0.5 mL/g, and 3.71 ± 0.5 mL/g; those of PGMA-EDMA-PEI and PGMA-EDMA by CRP were 210 ± 5 nm, 234 ± 5 nm, 1.61 ± 0.5 mL/g, and 1.78 ± 0.5 mL/g. The results indicated the pore size and pore volume were slightly reduced after modification of PGMA-EDMA microspheres. Both of the average pore sizes are more than 200 nm and the original permeability of the microspheres could be preserved in PGMA-EDMA-PEI microspheres.
3.9. Separation of proteins Fig. 10 showed the separation of three mode proteins mixture (lysozyme, PI = 11.0; ovalbumin, PI = 4.5; bovine serum albumin, 308PI = 4.7) on PGMA-EDMA-PEI with gradient elution. The baseline separation of three proteins could be achieved and the order of proteins in chromatographic curve was consistent with anion exchange chromatography mechanism. The fraction of each peaks (a, b, c) was analyzed by SDS-PAGE (as shown in Fig. 10). A single band was observed in each lane of SDS-PAGE pattern (as shown in lane a, b, and c) and indicated the three proteins had been well separated on the resin.
3.7. Protein binding capacity
4. Conclusion
The static and dynamic protein binding capacities of two supports were shown in Table 3. The corresponding front analysis chromatographic curves were shown in Fig. 7. The static binding capacity (Qs) of PGMA-EDMA-PEI (AGET ATRP) was two times of that on PGMA-EDMAPEI (CRP). This difference in QS was predictable due to higher surface (59.3 ± 1 m2/g) of PGMA-EDMA (AGET ATRP) than that (37.5 ± 1 m2/g) of PGMA-EDMA (CRP). Table 3 showed that dynamic binding capacities (QD) (at 3610 cm/h) on PGMA-EDMA-PEI (AGET ATRP) only decreased less than 10% in compared with that at 361 cm/ h. This value on PGMA-EDMA-PEI (CRP) reduced to be 11 ± 0.5 mg/ mL, which meant that QD decreased near half of that at 361 cm/h. As shown in Fig. 7, the 5% breakthrough values were reduced with flow rates. PGMA-EDMA-PEI by AGET ATRP (Fig. 7A) showed a slight decrease while resins by CRP significantly decreased. It was generally received that QD at different flow rates could indicate the mass transfer of proteins in the supports. QD at high flow rate differing little from one at low flow rate, as was reported in monolithic supports [30], meant that proteins could rapidly transfer in the microspheres by AGET ATRP. Meanwhile, the good interconnectivity of pores in the microspheres improved the availability of ion groups in the microspheres, which should be an attribution factor of protein binding capacity [31]. The pore distribution of microspheres in wet station were determined by ISEC and shown in Fig. 8. All of kd on PGMA-EDMA by CRP was in the range of 0.6–0.7. In other words, there was not obvious difference in Kd between dextran1500 (0.88 nm) and dextran3800000 (39.78 nm). This indicated that most of pores in PGMA-EDMA by CRP were more than 39.78 nm, which was in agreement with the above opinion of larger cluster forming the bigger pores. Kd on microspheres by AGET ATRP was in the range of 0.11–0.95 that was wider than that on microspheres by CRP. This demonstrated that PGMA-EDMA by AGET ATRP afforded the broad pore distribution and the high interconnectivity. Therefore, this microsphere had high surface area.
AGET ATRP suspension polymerization was firstly used to prepare macroporous microspheres, based on a copolymer of glycidyl methacrylate and ethylene dimethacrylate. In analogy to a conventional radical polymerization (CRP), microspheres by AGET ATRP afforded 10–300 nm particles which were smaller than CRP (400–800 nm particles). Therefore, microspheres by AGET ATRP showed high surface area that reached 59.3 ± 1 m2/g. It was worthy concerning that this method could gave both large pores (275 ± 5 nm) and high surface (59.3 ± 1 m2/g). The protein binding capacity on PGMA-EDMA-PEI by AGET ATRP was two folds of that on PGMA-EDMA-PEI by CRP. PGMAEDMA-PEI by AGET ATRP showed better mass transfer for proteins. These results indicated that the difference in polymerization mechanism between AGET ATRP and CRP could lead to different structure of polymer microspheres. What caused these differences need to be detailed studied in further research. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment The authors express thanks for the financial support from Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality (IDHT20180508), Beijing Natural Science Foundation (2162013, 2172054), and Construction of Scientific Research Platform (2018XK002). References
3.8. Adsorption of HBsAg in PGMA-EDMA-PEI
[1] R.S. Maier, M.R. Schure, Transport properties and size exclusion effects in widepore superficially porous particles, Chem. Eng. Sci. 185 (2018) 243–255. [2] Y. Li, F. Gao, W. Wei, J.B. Qu, G.H. Ma, W.Q. Zhou, Pore size of macroporous polystyrene microspheres affects lipase immobilization, J. Mol. Catal. B Enzym. 66 (2010) 182–189. [3] F. Svec, J.M.J. Fréchet, Kinetic control of pore formation in macroporous polymers. Formation of molded porous materials with high flow characteristics for separations or catalysis, Chem. Mater. 7 (1995) 707–715. [4] X. Zhou, B. Xue, S. Bai, Y. Sun, Macroporous polymeric ion exchanger of high capacity for protein adsorption, Biochem. Eng. J. 11 (2002) 13–17. [5] R. Zhang, Q. Li, Y. Gao, J. Li, Y. Huang, C. Song, W. Zhou, G. Ma, Z. Su, Hydrophilic modification of gigaporous resins with poly(ethylenimine) for high-throughput proteins ion-exchange chromatography, J. Chromatogr. A 1343 (2014) 109–118. [6] S. Li, Y. Sun, Q. Shi, Fabrication of high-capacity protein ion-exchangers with polymeric ion-exchange groups grafted onto micron-sized beads by atom transfer radical polymerization, Biochem. Eng. J. 103 (2015) 122–129. [7] E. Unsal, B. Elmas, B. Caglayan, M. Tuncel, S. Patir, A. Tuncel, Preparation of an ion-exchange chromatographic support by a grafting from strategy based on atom transfer radical polymerization, Anal. Chem. 78 (2006) 5868–5875. [8] S.D. Chambers, F. Svec, J.M.J. Fréchet, Incorporation of carbon nanotubes in porous polymer monolithic capillary columns to enhance the chromatographic separation of small molecules, J. Chromatogr. A 1218 (2011) 2546–2552. [9] A. Rahman, M. Iqbal, F. Rahman, D. Fu, M. Yaseen, Y. Lv, M. Omer, M. Garver, L. Yang, T. Tan, Synthesis and characterization of reactive macroporous poly(glycidyl methacrylate-triallyl isocyanurate-ethylene glycol dimethacrylate)
Considering the throughput pores and good mass transport, these microspheres by AGET ATRP should had better mass transfer for biomolecule with large size than those by CRP. In order to verify this point, hepatitis B surface antigens (HBsAg) with 22 nm size [32] marked by FI-TC were adsorbed in PGMA-EDMA-PEI and DEAE-Sepharose FF microspheres. These microspheres with HBsAg were observed by laser scanning confocal microscope (LSCM) and shown in Fig. 9. Fig. 9A1 (PGMA-EDMA-PEI) and A2 gave higher fluorescence intensity than Fig. 9B1 and B2 (DEAE-Sepharose FF). This indicated that HBsAg could diffuse into the interior of microspheres and be bound by the inner sites of ion-exchange due to throughput pores in PGMA-EDMA-PEI. However, DEAE-Sepharose FF only allowed HBsAg to be adsorbed on the surface of microspheres due to its small pore size (20–30 nm) [33]. Therefore, the higher capacity of proteins could be obtained on PGMAEDMA-PEI than DEAE-Sepharose FF. Meanwhile, it was observed that proteins with large size could better transport in PGMA-EDMA-PEI than DEAE-Sepharose FF due to the throughput pores in PGMA-EDMA-PEI. This similar result was also observed by Su [31]. 9
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[10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]
microspheres by suspension polymerization: effect of synthesis variables on surface area and porosity, J. Appl. Polym. Sci. 124 (2012) 36–47. E. Erbay, O. Okay, Pore memory of macroporous styrene-divinylbenzene copolymers, J. Appl. Polym. Sci. 71 (1999) 1055–1062. J.W. Kim, J.H. Ryu, K.D. Suh, Monodisperse micron-sized macroporous poly (styrene-co-divinylbenzene) particles by seeded polymerization, Colloid Polym. Sci. 279 (2001) 146–152. P.G. Gennes, Scaling Concept of Polymer Physics, Cornell University Press, 1979, p. 115. D. Horak, K. Labsky, J. Pilar, M. Bleha, Z. Pelzbauer, F. Svec, The effect of polymeric porogen on the properties of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate), Polymer 34 (1993) 3481–3489. F. Svec, Preparation and HPLC applications of rigid macroporous organic polymer monoliths, J. Sep. Sci. 27 (2004) 747–766. R. Zhang, L. Qi, P. Xin, G. Yang, Y. Chen, Preparation of macroporous monolith with three dimensional bicontinuous skeleton structure by atom transfer radical polymerization for HPLC, Polymer 51 (2010) 1703–1708. A.R. Wang, S. Zhu, Control of the polymer molecular weight in atom transfer radical polymerization with branching/crosslinking, J. Polym. Sci. A Polym. Chem. 43 (2005) 5710–5714. Q. Yu, F. Zeng, S. Zhu, Atom transfer radical polymerization of poly(ethylene glycol) dimethacrylate, Macromolecules 34 (2001) 1612–1618. P. Krys, K. Matyjaszewski, Kinetics of atom transfer radical polymerization, Eur. Polym. J. 89 (2017) 482–523. L. Yu, S. Tao, X. Dong, Y. Sun, Protein adsorption to poly(ethylenimine)-modified Sepharose FF: I. A critical ionic capacity for drastically enhanced capacity and uptake kinetics, J. Chromatogr. A 1305 (2013) 76–84. L. Hagel, P.L. Dubin (Eds.), Aqueous Size-Exclusion Chromatography, Elsevier, Amsterdam, 1988, p. 119. D. Lan, L. Bai, X. Pang, H. Liu, H. Yan, H. Guo, In situ synthesis of a monolithic material with multi-sized pores and its chromatographic properties for the separation of intact proteins from human plasma, Talanta 194 (2019) 406–414. T. Zhang, F. Zhou, J. Huang, R. Man, Ethylene glycol dimethacrylate modified hyper-cross-linked resins: porogen effect on pore structure and adsorption
performance, Chem. Eng. J. 339 (2018) 278–287. [23] J. Koubková, P. Müller, H. Hlídková, Z. Plichta, V. Proks, B. Vojtěšek, D. Horák, Magnetic poly(glycidyl methacrylate) microspheres for protein capture, New Biotechnol. 31 (2014) 482–491. [24] B. Gong, J. Zhu, L. Li, K. Qiang, L. Ren, Synthesis of non-porous poly(glycidylmethacryl- ate-co-ethylenedimethacrylate) beads and their application in separation of biopolymers, Talanta 68 (2006) 666–672. [25] O. Okay, Macroporous copolymer network, Prog. Polym. Sci. 25 (2000) 711–779. [26] K. Kanamori, J. Hasegawa, K. Nakanishi, T. Hanada, Facile synthesis of macroporous cross-linked methacrylate gels by atom transfer radical polymerization, Macromolecules 41 (2008) 7186–7193. [27] Y.Y. Chiu, L.J. Lee, Microgel formation in the free radical crosslinking polymerization of ethylene glycol dimethacrylate (EGDMA). I. Experimental, J. Polym. Sci. A Polym. Chem. 33 (1995) 257–267. [28] O. Okay, Phase separation in free-radical crosslinking copolymerization: formation of heterogeneous polymer networks, Polymer 40 (1999) 4117–4129. [29] R. Zhang, Q. Li, J. Li, W. Zhou, P. Ye, Y. Gao, G. Ma, Z. Su, Covalently coating dextran on macroporous polyglycidyl methacrylate microsphere enabled rapid protein chromatographic separation, Mater. Sci. Eng. C 32 (2012) 2628–2633. [30] M. Wang, J. Xu, X. Zhou, T. Tan, Preparation and characterization of polyethyleneimine modified ion-exchanger based on poly(methacrylate-co- ethylene dimethacrylate) monolith, J. Chromatogr. A 1147 (2007) 24–29. [31] M. Yu, Y. Li, S. Zhang, X. Li, Y. Yang, Y. Chen, G. Ma, Z. Su, Improving stability of virus-like particles by ion-exchange chromatographic supports with large pore size: advantages of gigaporous media beyond enhanced binding capacity, J. Chromatogr. A 1331 (2014) 69–79. [32] Z. Kimia, S.N. Hosseini, S.S.A. Talesh, M. Khatami, A. Kavianpour, A. Javidanbardan, A novel application of ion exchange chromatography in recombinant hepatitis B vaccine downstream processing: improving recombinant HBsAg homogeneity by removing associated aggregates, J. Chromatogr. B 1113 (2019) 20–29. [33] A. Jungbauer, Chromatographic media for bioseparation, J. Chromatogr. A 1065 (2005) 3–12.
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Fabrication of poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) macroporous microspheres through activators regenerated by electron transfer atom transfer radical polymerization for rapid separation of proteins
T
Ning Daib,1, Shaoyun Wangd,1, Heng Lia, Lan Zhaoc, Haibo Jina, Ning Ana, Pisheng Gonga, ⁎ Qiqi Tana, Xu Tanga, Fei Wanga, Rongyue Zhanga, a
Beijing Key Laboratory of Fuels Cleaning and Advanced Catalytic Emission Reduction Technology/College of Chemical Engineering, Beijing Institute of Petrochemical Technology, Beijing 102617, China Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China c National Key Lab of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China d Senhui Microsphere Tech (Suzhou) Co., Ltd., Suzhou 215123, China b
ARTICLE INFO
ABSTRACT
Keywords: Macroporous microspheres Suspension polymerization Atom transfer radical polymerization Separation of proteins
Activators regenerated by electron transfer atom transfer radical polymerization (AGET ATRP) were firstly used in suspension polymerization to prepare macroporous microspheres based on a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate. Compared to conventional radical polymerization (CRP), the microspheres by AGET ATRP showed more homogeneous structure, larger pores, and higher protein binding capacity. The body of microspheres are formed by the large clusters resulted from the aggregated little particles. The size of the particles in microspheres by AGET ATRP was 10–300 nm which was smaller than that (400–800 nm) of the microspheres by CRP. AGET ATRP gave larger pore size (275 ± 5 nm) and surface area (59.3 ± 1 m2/g) than CRP (234 ± 5 nm, 37.5 ± 1 m2/g). The microspheres were modified with polyethylene imine for anion resins that were evaluated in term of its protein binding capacity. The results indicated that the static (69 ± 0.5 mg/mL) and dynamic binding capacity (61 ± 0.5 mg/mL) of proteins on modified microspheres by AGET ATRP were higher than that (34 ± 0.5 mg/mL and 19 ± 0.5 mg/mL) by CRP. Meanwhile, the proteins binding capacity on the microspheres by AGET ATRP decreased only less than 10% when the flow rate increased 10 times. These macroporous media show a large potential in rapid separation of proteins.
1. Introduction Polymer microspheres, as an attractive chromatographic medium, have been widely used for separation and purification of proteins due to its good mechanical strength and chemical stability. Generally, the large pores of the media play a major role in realizing high throughput chromatography. It is well known that the large pore size is advantageous in separation for rapid mass transport [1,2]. However, the larger pores usually cause the smaller surface areas in the porous microspheres. The surface areas of the macroporous microspheres will decrease to less than 60 m2/g when its average pore size is more than 100 nm, and even be reduced to less than 10 m2/g [3]. As a result, low protein binding capacity is often observed on the chromatographic
media with large pores [4]. Therefore, it has been an attractive topic for researchers to improve the protein binding capacity on the chromatography with large pores. As a common method, grafting polymers in these media was usually used for increasing the capacity. In our previous study [5], coating branched polyethyleneimines (PEI) on the gigaporous microspheres (based on a copolymer of glycidyl methacrylate and divinyl benzene, PGMA-DVB) was conducted and the protein binding capacity had a rising trend with the amount of PEI in the range of 0.11–0.24 mmol/mL ion exchange capacity. Meanwhile, it was found that the capacity began to decrease when the ion exchange capacity was more than 0.24 mmol/ mL. The possible reason was that extra PEI chains reduced surface area of microspheres, so, the adsorption sites on the PEI were buried and
Corresponding author. E-mail address: [email protected] (R. Zhang). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.jchromb.2019.121794 Received 5 June 2019; Received in revised form 11 August 2019; Accepted 7 September 2019 Available online 10 September 2019 1570-0232/ © 2019 Elsevier B.V. All rights reserved.
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could not bind proteins. In reported methods, it also was found that the protein binding capacity reached a constant value or began to decrease when the amount of grafting polymers exceeded a certain value [6,7]. As an alternative way, Svec's group [8] used nanoparticles to coat the polymer monolith for increasing its surface area, and a high column efficiency was obtained in this modified monolith while the reducing pore size increased column pressures. The above-mentioned methods could be summarized as the same approach, in which the protein binding capacity could be improved through modifying the surface of the macroporous matrix with multifunctional compounds to increase adsorption sites. In this way, the structure of the media did not change except for slightly reducing the pore sizes due to the modifying compounds occupying some volume in the media. However, capacity was limitedly promoted owing to the reduced surface area of the matrix. Therefore, fabrication of macroporous supports with high surface area should be an alternative method for increasing protein capacity without sacrificing large pores. The macroporous microspheres are generally prepared though suspension polymerization initiated by conventional radical polymerization (CRP) [9]. In this method, polymerization takes place in each emulsion drop and the formation of pores depends on the process of phase separation between polymers and porogens. Therefore, screening reaction conditions and porogens are always concerned by many researchers [10,11] for large pores, but the matrix structure of the microspheres is not remarkably changed due to the nature of CRP. In this way, the growing polymer chains in CRP tend to aggregate each other because van der Waals attraction surmounts the steric hindrance mutually expelling the polymer chains [12], and the segregated polymer chains soon form tiny particles, then those particles aggregate to the typical heterogeneous macroporous structures composed of micron size global particles [13,14]. The micron size particles and random aggregation led to low surface area and heterogeneous structure of the supports. To address this problem, we ever used atom transfer radical polymerization (ATRP) to fabricate the monolith and found that ATRP could get higher surface area and more homogenous structure [15] than CRP. It is also proved by polymer gels prepared through ATRP that are more homogeneous than ones by CRP [16]. In comparison with CRP, the concentration of propagating radicals is low during polymerization in ATRP [17]. Hence, it often takes hours for an individual chain to propagate hundreds of monomer units, and thus chains growth through propagation is slowed down [18]. In contrast to this, only seconds for the same number of monomer units are needed to be added to the polymer chain in CRP while the process of chain relaxation would need minutes or hours. Therefore, the larger micro particles would be formed in CRP than those in ATRP and resulted in low surface area and heterogeneous structure. Based on the above description, the macroporous microspheres are expected to afford a high surface area and protein binding capacity through suspension polymerization initiated by ATRP. The aim of this study is to explore suspension ATRP and fabricate macroporous microspheres with great surface area, large pores, and high protein binding capacity.
Dichloromethane (DCM), n-octanol (OA), poly(vinyl alcohol) (PVA, Mw = 1700 ± 50, 88% alcoholysis degree), sodium lauryl sulfate (SLS), ascorbic acid (AC) and CuBr2 were purchased from Beijing Chemical Plant (Beijing, China). Poly(ethylene imine) (PEI, Mw = 600) was obtained from XIYA company (Shandong, China). Lysozyme, ovalbumin, proteins marker, and bovine serum albumin (BSA) was purchased from Sigma–Aldrich (St. Louis, MO, USA). All the above chemicals were received. Other reagents used in experiments were of analytical grade and bought from Beijing Chemical Factory (Beijing, China). 2.2. Preparation of PGMA-EDMA microspheres by AGET ATRP In a typical polymerization, GMA (5.0 mL, 38.5 mmol), EDMA (5.0 mL, 26.5 mmol), EBP (0.017 mL, 1.3 mmol), CuBr2 (0.29 g, 1.3 mmol), DCM (15.0 mL) and OA (15.0 mL) were added to an ampule. The ampule was then sealed with a rubber septum and degassed with nitrogen for 20 min. Deoxidized PMDETA (0.27 mL, 1.3 mmol) was quickly added to the ampule by a degassed syringe. The ampule was shaken for 30 s and the reaction mixture was subsequently injected into the reactor containing 160 mL aqueous solution composed by 4.8 g PVA, 1.5 g ascorbic acid and 0.16 g SLS under 210 rpm stirring. The reactor was heated to be 60 °C and the polymerization proceeded at 60 °C for 8 h. The reaction solution was filtered and successively washed with deionized water and ethanol; the resulted microspheres were then extracted in a Soxhlet apparatus with acetone and deionized water for 48 h, respectively. Finally, the microspheres were stored in 20% ethanol aqueous solution at room temperature. As a parallel experiment, the same processes were performed by conventional radical polymerization initiated by AIBN. In these comparative experiments, all of reaction conditions and the following treatments were as the same as AGET ATRP except for no CuBr2 added in these reaction systems. 2.3. Modification of PGMA-EDMA microspheres with PEI
2. Material and methods
In order to measure the protein binding capacity on the PGMAEDMA microspheres, PEI was used to modify the supports for anion exchange media. This derivation was carried out according to the previous study [5]. The typical procedure was shown in Scheme 1 and began with copolymerization of GMA and EDMA by AGET ATRP at 60 °C for 8 h, followed by attaching PEI with required molecules to PGMA-EDMA microspheres in dimethyl sulfoxide (DMSO) for 24 h at 60 °C. The ionic capacity of modified microspheres (PGMA-EDMA-PEI) was determined by chloride-silver nitrate titration as described by Sun et al. [19]. The modified microspheres (10 mL) were filled into a glass column, and then was washed with 10 column volumes (CVs) of 1.0 mol/L NaCl to saturate the ion exchange sites with chloride ions and rinsed with 0.1 mmol/L HCl. Then, the column was rinsed with five CVs of Na2SO4 (10%, w/w), during which the bound chloride ions were replaced with sulfates. Chloride ions in the effluent were titrated with 50 mmol/L AgNO3 in the presence of K2CrO4 (0.5 mL, 5%, w/w) as an indicator. Then, the IC of the column was calculated from the titration data.
2.1. Materials
2.4. Pores distribution and morphology of the microspheres in dry state
Glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) were purchased from Sigma–Aldrich (St. Louis, MO, USA) and purified before use. Ten dextran standards of molecular weights ranging from 342 to 2,285,000 Da were obtained from Sigma–Aldrich (St. Louis, MO, USA). Azobisisobutyronitrile (AIBN) was produced by Shanghai Chemical Plant (Shanghai, China) and refined by recrystallization from the methanol before use. N,N,N′,N″,N″-pentamethyl diethylenetriamine (PMDETA) was purchased from JK Chemical Ltd. (Beijing, China). Ethyl-2-bromopropionate (EBP) was obtained from Alfa Aesar (Lancs, England). DEAE-Sepharose fast flow was from GE company (USA).
Pore size distribution of PGMA-EDMA by two methods, including AGET ATRP and CRP, as well as PGMA-EDMA-PEI, in their dry state, were determined by an Auto Pore IV 9500 mercury porosimeter (Micromeritics, USA). The specific surface area and porosity of the samples were measured by N2 adsorption on a Micromeritics ASAP2020 analyzer (Micromeritics, USA) and calculated by the Brunauer-EmmettTeller (BET) method. The morphologies of these microspheres were observed by scanning electron microscope (SEM) (JSM-6400, JEOL Lim. Co., Japan). The detailed method of sample preparations for SEM observation was described as following. Microspheres were re2
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Scheme 1. Preparation and modification of PGMA-EDMA microspheres.
suspended in distilled water. Then, the dispersion was dropped on a piece of aluminum foil and dried at an ambient atmosphere. The sample was placed on a metal stub with double-sided conductive adhesive tape, and then it was coated with a thin gold film under reduced pressure below 5 Pa with a JFC-1600 fine coater (JEOL, Japan).
2.6. Determination of static and dynamic binding capacities Static binding capacity (SBC) experiments were carried out in the equilibration buffer (20 mmol/L Tris-HCl buffer, pH 8.0) using the method in the previous report [5]. The typical procedure was performed as the following: 0.1 g microspheres was firstly equilibrated with the buffer and then was mixed with 10 mL of protein solution (BSA, 0.3–3.0 mg/mL) prepared in the same buffer and incubated at 120 rpm and 21 °C for 24 h. The supernatant was subsequently collected by centrifuging at 5000 rpm for 5 min and protein concentration (c) in the supernatant was finally measured at 280 nm using a UV transparent 384 well plate (Brand, Wertheim, Germany) and the adsorption capacity of protein (qS) was calculated by mass balance,
2.5. Measurement of pore distribution of the microspheres in wet state by inversed size exclusive chromatography (ISEC) The microspheres were packed into the columns (ø10 × 500 mm) and the final packed bed height was 50 cm. The parameters of the dextran, such as the weight-average molecular weight Mw, the numberaverage molecular weight Mn, the molecular weight corresponding to the elution peak Mp, the viscosity radius Rη, and the polydispersity (Mw/Mn), were shown in Table 1 [20]. Dextran solutions were prepared by dissolution in the operating buffer (200 mM NaCl, 20 mM Tris-HCl solution, at pH 7.0) and filtered through 0.22 μm membrane to remove possible aggregates prior to injection into the columns. Waters 2695 equipped with 2414 refractive index and 2696 photodiode array detector (Waters, Milford, MA, USA) was used to determined chromatography behaviors of the microspheres at room temperature. ISEC was performed at 1.0 mL/min, using injection volumes of 100 μL. The interparticle volume V0 was determined using monodisperse nano-SiO2 particles (80 nm) as the excluded solute. The total mobile phase volume VT was measured using glucose. The SEC distribution coefficient, Kd, was measured by the following equation:
Kd =
Ve
V0 Vp
=
Ve VT
V0 V0
qS =
qD =
Mna
Mw/Mna
Rη (nm)b
1500 5000 12,000 25,000 50,000 80,000 150,000 270,000 410,000 670,000 3,800,000
1080 4440 9890 21,400 43,500 66,700 123,600 196,300 276,500 401,300 2,285,000
1350 5220 11,600 23,800 48,600 80,900 147,600 273,000 409,800 667,800 3,790,000
1160 3260 8110 18,300 35,600 55,500 100,300 164,200 236,300 332,800 1,500,000
1.16 1.6 1.43 1.30 1.36 1.46 1.47 1.66 1.73 2.01 2.53
0.88 1.78 2.65 3.89 5.53 6.845 9.31 11.72 13.90 16.73 39.78
a b
V0 ) × c V
(3)
2.7. Transport of proteins in the microspheres by CLSM The transport of labeled proteins in the microspheres was monitored by TCS SP2 confocal laser scanning microscope (CLSM) (Leica, Germany). Fluorescein isothiocyanate-labeling hepatitis B surface antigen (FITC-HBsAg) adsorption experiments were conducted in a 4 °C incubator, after 12 h adsorption, the microspheres were separated from the FITC-HBsAg solution by centrifugation and washed with 50 mM Tris-HCl buffer three times to remove desorbed protein molecules completely. After being placed on a slide glass and being covered with cover glass, the microspheres were observed with LCSM to visualize the adsorption of FITC-HBsAg onto microspheres. The samples were detected at 488 nm, and the fluorescent images at 520–550 nm wavelengths were then taken.
Table 1 Molecular weights (Mp, Mw, Mn), polydispersity (Mw/Mn) and viscosity radii (Rη) of the dextran standards used in ISEC. Mwa
(V1
where V1 is the 5% breakthrough volume, V0 is the sum of void volumes of column and HPLC system, c is the concentration of protein (mg/mL), and V is the volume of media in column (mL).
where Ve was the elution volume of the analyte and Vp was interparticle volume.
Mpa
(2)
where qm is the maximum adsorption capacity and K is the Langmuir dissociation constant. The isothermal data was fitted using Sigma Plot 8 (Systat Software Inc., San Jose, CA, USA). Dynamic binding capacity (DBC) was determined by frontal analysis experiments according to the reported method [5]. Frontal analysis experiments were performed at pH 8.0 with 1.0 mg/mL BSA in pH 8.0 Tris-HCl buffer. The dynamic binding capacity (qD) was calculated by Eq. (3)
(1)
Dextran standard
qm × c K+c
2.8. Separation of proteins Separation of model proteins mixture (including lysozyme, ovalbumin, bovine serum albumin) processes were carried out using 20 mM
All of data was from quality inspection of productions. Rη were calculated using the correlation Rη = 0.0271×Mp0.498 [20]. 3
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3.2. Effect of polymerization temperature on morphology of microspheres
Tris-HCl pH 8.0 as the running buffer A and 20 mM Tris-HCl 1 M NaCl pH 8.0 as the elution buffer B. PGMA-EDMA-PEI was packed into a column (Ф10 × 300 mm) with a final bed height of 25.4 cm and a bed volume of 20 mL. Before loading, the column was equilibrated for 5 column volume (CV) with buffer A. After loading, the column was washed with buffer A for 2.5 CV and then eluted by a linear gradient elution to 100% buffer B with a gradient length of 12 CV at the flow rate of 5 mL/min. Fractions of the flow-through as well as the eluates were collected and further analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyacrylamide-based gel was prepared according to manufacturer's instructions using Liu Yi DYCZ-28B electrophoresis (Beijing Liu Yi, Beijing, China). After completion of running the gel at 100 V, the gel was removed from the tank and stained with Coomassie dye (Thermo Fisher Scientific, Waltham, MA, USA). The gel was washed with DDI water until all protein bands were clearly observed. SDS-PAGE images were obtained by camera.
Polymerization temperature is often used for controlling the pore size distribution in polymer porous materials. In convention radical polymerization (CRP), the lower the polymerization temperature, the larger the pores [3]. Keeping other condition fixed (1:1 GMA/EDMA ratio, 3:1 porogen/monomers ratio, 1:1 DCM/OA ratio), the temperature varied from 55 to 70 °C. Because the boiling point of DCM is 39.8 °C and will be lost in high temperature, more than 70 °C is not be considered in this experiment. Fig. 2 shows the effect of temperature on the morphology of microspheres prepared at 55 °C (A1, C1), 60 °C (A2, C2), 65 °C (A3,C3) and 70 °C (A4, C4). In comparison to CRP (Fig. 2 C14), AGET ATRP (Fig. 2 A1-4) showed a similar trend of decrease of pores size with temperature, but no significant changes of morphology were observed on the microspheres by AGET ATRP, and the interconnected network structure was still retained. On the contrast, CRP showed that the size of aggregation particles had a great decrease with temperature (Fig. 2C1-4). Meanwhile, no interconnected network in microspheres by CRP can be found at each temperature. As indicated above, it was found the pore size and pores interconnectivity decreased with reaction temperature. Generally, the large pore and high interconnectivity were the key factors for realizing fast transport of proteins. The formation of microspheres that consumed less than 10 min was faster than CRP (4 h) at 60 °C. In order to obtain high throughput matrix, 60 °C is considered for the optimal polymerization temperature.
3. Results and discussion 3.1. Effect of GMA to EDMA ratio on morphology of microspheres GMA to EDMA ratio had an influence on the morphology of microspheres [21]. The polymerization was conducted in the range from 2:1 to 0.5:1 GMA/EDMA ratio, and the others conditions were constant in all polymerization, including porogen to monomers ratio (3:1, v/v), porogenic solvent (1:1 (v/v) dichloromethane (DCM) /n-octanol (OA) ratio), temperature (60 °C) and reaction time (8 h). In order to compare CRP and AGET ATRP, the same polymerizations conditions in AGET ATRP were carried out as those in CRP with AIBN as initiator. As shown in Fig. 1, Fig. 1 A1-A4 were the morphology of microspheres from AGET ATRP and the size of aggregation particles was reduced with the decrease of GMA/EDMA ratio. Meanwhile, the network of microspheres became more homogenous. In microspheres by CRP, the similar trend was also observed (Fig. 1 C1-C4), but the heterogenous structure was higher than microspheres by AGET ATRP at each reaction condition. Although, the low GMA/EDMA ratio can afford homogeneous network, and a low GMA would decrease the content of reactive epoxide groups that are needed for the subsequent functionalization of the microspheres. To obtain microspheres with homogenous structure and enough reactive epoxide groups, the 1:1 GMA/EDMA ratio is selected for the optimal ratio.
3.3. The effect of porogen to monomers ratio on the morphology All of both type of microsphere from AGET ATRP (A1-3) and CRP (C1-3) afford increased pores size and pore volume with the porogen/ monomer ratio. Generally, the pores of the microspheres are from the voids within the porous beads and the fraction of voids is close to the volume fraction of the porogen in the initial polymerization mixture. Compared to the effect of GMA/EDMA and temperature, the porogen/ monomer ratio had significant influence on microspheres by AGET ATRP [22–24]. As might be expected, microspheres by AGET ATRP (Fig. 3A1-3) gave more pores and better interconnected pores in comparison to CRP (Fig. 3. These features of microspheres by AGET ATRP are advantageous in promoting rapid mass transfer through a porous microsphere. Meanwhile, the large pore size and high surface area can be expected in these porous microspheres. The polymerization system
Fig. 1. SEM of microspheres at different GMA/EDMA ratio, A1-4:2:1, 1.5:1, 1:1, 0.5:1, by AGET ATRP; C1-4: 4:2:1, 1.5:1, 1:1, 0.5:1, by CRP. 4
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Fig. 2. SEM of microspheres at various temperatures, A1-4: 55, 60, 65, 70 °C, by AGET ATRP; C1-4, 55, 60, 65, 70 °C, by CRP.
was designed according to the conditions, including 1:1 GMA/EDMA, 3:1 porogen/monomers, 1:1 DCM/OA ratio, 60 °C and 8 h. All of microspheres in the following study, as the matrix, is prepared by this polymerization condition if no other description is cited.
Table 2 The structure parameters of microspheres by mercury intrusion.
3.4. Comparison of microspheres by AGET ATRP and CRP
Supporta
Total intrusion volumeb (mL/g)
Average pore diameter (nm)b
Surface area (m2/g)c
Porosity (%)b
AGET ATRP CRP
3.71 ± 0.5 1.78 ± 0.5
275 ± 5 234 ± 5
59.3 ± 1 37.5 ± 1
88.5 ± 2 69.8 ± 2
a All of microspheres was prepared by the same reaction conditions: 1:1 GMA/EDMA, 3:1 porogen/monomers, 1:1 DCM/OA ratio, 60 °C and 8 h. b These data were measured by mercury intrusion method. c These data were determined by N2 adsorption method.
Based on the optimized polymerization conditions, the microsphere structures, including pore size, surface area, porosity, and morphology, were compared between AGET ATRP and CRP. The relative results were shown in Table 2 and Fig. 4, respectively. Compared to CRP, all of structure parameters of microspheres by AGET ATRP, including average
Fig. 3. SEM of microspheres at various porogen/monomer ratio, A1-3: 1:1, 2:1, 3:1, by AGET ATRP; C1-3, 1:1, 2:1, 3:1, by CRP. 5
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Fig. 4. SEM of microspheres by AGET ATRP (1) and CRP (2).
Scheme 2. Gel formation in the droplets by two ways of AGET ATRP and CRP.
Fig. 5. The backpressures on PGMA-EDMA at various flow rates.
Fig. 6. Pore distributions of microspheres by mercury intrusion.
pore diameter, surface area, and porosity, was higher. Especially, both the average pore and surface area by AGET ATRP (275 ± 5 nm, 59.3 ± 1 m2/g) were higher than those from CRP (234 ± 5 nm, 37.5 ± 1 m2/g). The usual rule was well known that the larger pore size usually caused the lower surface area [25]. However, it was found that the surface area of the microspheres by AGET ATRP (275 nm pore size) was more 58% than CRP with 234 nm pore size. This abnormal result aroused us interest in exploring the structure difference between these two microspheres by AGET ATRP and CRP. The morphologies and of PGMA-EDMA by AGET ATRP and CRP
were observed through scanning electronic microscopy (SEM) and shown in Fig. 4. In Fig. 4, Fig. 4-1 (AGET ATRP) showed that the polymer skeleton was composed by hundred-nanometer particles (10–300 nm) that was smaller than CRP (400–800 nm, Fig. 4-2). Meanwhile, higher porosity and inter-connectivity could be observed on the Fig. 4-1 than those from PGMA-EDMA by CRP (Fig. 4-2). Generally, the small particles or clusters bring large surface area [25]. PGMA-EDMA by AGET ATRP therefore showed higher surface area than CRP. According to the general rule that larger particles or clusters give larger pores, microspheres by CRP could have afforded larger pores 6
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Table 3 Static and dynamic binding capacity of PGMA-EDMA-PEI microspheres. Support
QS (mg/mL)
a
QD (mg/mL)
b
QD (mg/mL)
c
QD (mg/mL)
d
PGMA-EDMA-PEI (AGET ATRP) PGMA-EDMA-PEI (CRP)
69 ± 0.5
61 ± 0.5
59 ± 0.5
58 ± 0.5
56 ± 0.5
34 ± 0.5
19 ± 0.5
14 ± 0.5
13 ± 0.5
11 ± 0.5
QD (mg/mL)
a,b,c,d
The dynamic binding capacities were determined at the flow rate of 361 cm/h, 1444 cm/h, 2527 cm/h, and 3610 cm/h, respectively.
than AGET ATRP. On the contrary, microspheres by AGET ATRP showed larger pores than CRP. The possible reason was that the high interconnectivity contributed to large pore and porosity in microspheres by AGET ATRP. These differences should be relative with way of polymerization. In our previous preparation of monolith by AGET ATRP, the obtained monolith showed three-dimension bicontinuous skeleton while the monolith by CRP was composed from aggregation of particles under the same conditions [15] as ATRP. Kanamori also reported the similar result and the monolith by living polymerization could obtain three dimensional bicontinuous skeleton structure [26]. The similar explanation for formation of this structure was that living polymerization gave more homogeneous gels than CRP. In present study, ATRP suspension polymerization was used to prepare microspheres, which differed from in-situ polymerization in effect of the tension at liquid-liquid interface [3] not in gel structure formation. In suspension polymerization, the gel is formed in each droplet that is considered as a reaction unit. The droplet, including reactive composition, porogen and initiator, formed the microsphere after polymerization. Meanwhile, each microsphere is also considered as a gel that is formed from cross linked microgels (tiny particles). The resulted structure of microsphere mainly depended on the microgel size and how to be crosslinked. The formations of gels by the two ways, ATRP and CRP, were described by Scheme 2. As shown in Scheme 2, the gel formation began with polymerization in a droplet, then many polymer chains with similar molecular weights were formed by ATRP and dispersed in the porogen solvent prior to crosslinking reaction (or in the early stage of polymerization), which was attributed to the nature of living/control polymerization (well controlling molecular weight of polymer); these polymer chains were followed by crosslinking reaction with further polymerization and homogenous gel was finally formed. In CRP, many micro gels with different sizes were developed in the early polymerization stage due to the nature of CRP [27,28]; the micro gels aggregated and formed larger gel particles (clusters) due to local fluctuation of micro gels concentration with polymerization. These
Fig. 8. Pore distributions of microspheres by ISEC.
particles (clusters) were crosslinked and resulted in the heterogeneity of the resulted gels. This heterogeneity led to low connectivity between the pores in the microspheres. 3.5. Permeability of PGMA-EDMA microspheres In the case of porous materials, high porosity and connectivity mean good permeability or high throughput for separation. To prove this point, the permeability of PGMA-EDMA was determined by evaluating their backpressures under different flow rates. PGMA-EDMA microspheres were filled in the stainless columns (ø10 × 50 mm). Fig. 5 showed that the backpressures of two columns had a linear increase with flow rates (in the range of 361–2889 cm/h), and this indicated that good mechanical strength could be kept under the testing pressures [29]. Meanwhile, PGMA-EDMA by AGET ATRP had lower back pressure than ones by CRP at each flow rate. This proved that PGMA-EDMA by AGET ATRP had better permeability than that by CRP. These comparisons further verified the observation from SEM, the pores in PGMAEDMA by AGET ATRP had better interconnectivity than those by CRP. 3.6. Modification of PGMA-EDMA microspheres by PEI From the above-mentioned results, it could be found that AGET ATRP could give higher surface area and permeability than CRP. Next, it was explored whether these microspheres by AGET ATRP could give
Fig. 7. Dynamic binding capacity on PGMA-EDMA-PEI at different flow rates, (A, microspheres by AGET ATRP, B, microspheres by CRP). 7
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Fig. 9. LSCM of HBsAg on PGMA-EDMA-PEI by AGET ATRP and CRP, A1 and A2, AGET ATRP; B1 and B2, CRP.
Fig. 10. Separation of proteins on PGMA-EDMA-PEI and SDS-PAGE of the corresponding chromatographic peak fractions. Peak a, lysozyme, b, ovalbumin, c, BSA; Lane a, b, c: the collected fraction of peak a, b, and c, respectively; Lane M, proteins marker.
higher protein binding capacity. To examine this, the microspheres were modified by PEI to obtain anion exchange supports. PEI was used for modification of gigaporous microspheres (poly(glycidyl methacrylate-co-divinyl benzene)) to separation of proteins in our previous study [5]. The ion capacities of modified PGMA-EDMA microspheres were
determined and the results were 0.21 mmol/mL (PGMA-EDMA by AGET ATRP) and 0.23 mmol/mL (PGMA-EDMA by CRP). The effect of modification on the microspheres structure was also an important item to be concerned in our study. The pore distribution of modified microspheres was determined by mercury intrusion method and shown in 8
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Fig. 6. The average pore and pore volume of PGMA-EDMA-PEI and PGMA-EDMA by AGET ATRP were 268 ± 5 nm, 275 ± 5 nm, 3.50 ± 0.5 mL/g, and 3.71 ± 0.5 mL/g; those of PGMA-EDMA-PEI and PGMA-EDMA by CRP were 210 ± 5 nm, 234 ± 5 nm, 1.61 ± 0.5 mL/g, and 1.78 ± 0.5 mL/g. The results indicated the pore size and pore volume were slightly reduced after modification of PGMA-EDMA microspheres. Both of the average pore sizes are more than 200 nm and the original permeability of the microspheres could be preserved in PGMA-EDMA-PEI microspheres.
3.9. Separation of proteins Fig. 10 showed the separation of three mode proteins mixture (lysozyme, PI = 11.0; ovalbumin, PI = 4.5; bovine serum albumin, 308PI = 4.7) on PGMA-EDMA-PEI with gradient elution. The baseline separation of three proteins could be achieved and the order of proteins in chromatographic curve was consistent with anion exchange chromatography mechanism. The fraction of each peaks (a, b, c) was analyzed by SDS-PAGE (as shown in Fig. 10). A single band was observed in each lane of SDS-PAGE pattern (as shown in lane a, b, and c) and indicated the three proteins had been well separated on the resin.
3.7. Protein binding capacity
4. Conclusion
The static and dynamic protein binding capacities of two supports were shown in Table 3. The corresponding front analysis chromatographic curves were shown in Fig. 7. The static binding capacity (Qs) of PGMA-EDMA-PEI (AGET ATRP) was two times of that on PGMA-EDMAPEI (CRP). This difference in QS was predictable due to higher surface (59.3 ± 1 m2/g) of PGMA-EDMA (AGET ATRP) than that (37.5 ± 1 m2/g) of PGMA-EDMA (CRP). Table 3 showed that dynamic binding capacities (QD) (at 3610 cm/h) on PGMA-EDMA-PEI (AGET ATRP) only decreased less than 10% in compared with that at 361 cm/ h. This value on PGMA-EDMA-PEI (CRP) reduced to be 11 ± 0.5 mg/ mL, which meant that QD decreased near half of that at 361 cm/h. As shown in Fig. 7, the 5% breakthrough values were reduced with flow rates. PGMA-EDMA-PEI by AGET ATRP (Fig. 7A) showed a slight decrease while resins by CRP significantly decreased. It was generally received that QD at different flow rates could indicate the mass transfer of proteins in the supports. QD at high flow rate differing little from one at low flow rate, as was reported in monolithic supports [30], meant that proteins could rapidly transfer in the microspheres by AGET ATRP. Meanwhile, the good interconnectivity of pores in the microspheres improved the availability of ion groups in the microspheres, which should be an attribution factor of protein binding capacity [31]. The pore distribution of microspheres in wet station were determined by ISEC and shown in Fig. 8. All of kd on PGMA-EDMA by CRP was in the range of 0.6–0.7. In other words, there was not obvious difference in Kd between dextran1500 (0.88 nm) and dextran3800000 (39.78 nm). This indicated that most of pores in PGMA-EDMA by CRP were more than 39.78 nm, which was in agreement with the above opinion of larger cluster forming the bigger pores. Kd on microspheres by AGET ATRP was in the range of 0.11–0.95 that was wider than that on microspheres by CRP. This demonstrated that PGMA-EDMA by AGET ATRP afforded the broad pore distribution and the high interconnectivity. Therefore, this microsphere had high surface area.
AGET ATRP suspension polymerization was firstly used to prepare macroporous microspheres, based on a copolymer of glycidyl methacrylate and ethylene dimethacrylate. In analogy to a conventional radical polymerization (CRP), microspheres by AGET ATRP afforded 10–300 nm particles which were smaller than CRP (400–800 nm particles). Therefore, microspheres by AGET ATRP showed high surface area that reached 59.3 ± 1 m2/g. It was worthy concerning that this method could gave both large pores (275 ± 5 nm) and high surface (59.3 ± 1 m2/g). The protein binding capacity on PGMA-EDMA-PEI by AGET ATRP was two folds of that on PGMA-EDMA-PEI by CRP. PGMAEDMA-PEI by AGET ATRP showed better mass transfer for proteins. These results indicated that the difference in polymerization mechanism between AGET ATRP and CRP could lead to different structure of polymer microspheres. What caused these differences need to be detailed studied in further research. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment The authors express thanks for the financial support from Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality (IDHT20180508), Beijing Natural Science Foundation (2162013, 2172054), and Construction of Scientific Research Platform (2018XK002). References
3.8. Adsorption of HBsAg in PGMA-EDMA-PEI
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Considering the throughput pores and good mass transport, these microspheres by AGET ATRP should had better mass transfer for biomolecule with large size than those by CRP. In order to verify this point, hepatitis B surface antigens (HBsAg) with 22 nm size [32] marked by FI-TC were adsorbed in PGMA-EDMA-PEI and DEAE-Sepharose FF microspheres. These microspheres with HBsAg were observed by laser scanning confocal microscope (LSCM) and shown in Fig. 9. Fig. 9A1 (PGMA-EDMA-PEI) and A2 gave higher fluorescence intensity than Fig. 9B1 and B2 (DEAE-Sepharose FF). This indicated that HBsAg could diffuse into the interior of microspheres and be bound by the inner sites of ion-exchange due to throughput pores in PGMA-EDMA-PEI. However, DEAE-Sepharose FF only allowed HBsAg to be adsorbed on the surface of microspheres due to its small pore size (20–30 nm) [33]. Therefore, the higher capacity of proteins could be obtained on PGMAEDMA-PEI than DEAE-Sepharose FF. Meanwhile, it was observed that proteins with large size could better transport in PGMA-EDMA-PEI than DEAE-Sepharose FF due to the throughput pores in PGMA-EDMA-PEI. This similar result was also observed by Su [31]. 9
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