Effect of Porogen Solubility Parameter on Structure of Chromatographic Supports with Large Pores

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 46, Issue 2, February 2018 Online English edition of the Chinese language journal

Cite this article as: Chinese J. Anal. Chem., 2018, 46(2): 288–292

RESEARCH PAPER

Effect of Porogen Solubility Parameter on Structure of Chromatographic Supports with Large Pores LAN Meng-Fei1, AN Ning1, ZHAO Ying1, CAO Wei1, LI Heng1, ZHAO Lan2, HUANG Yong-Dong2, ZHANG Rong-Yue1,* 1

Beijing Key Laboratory of Fuels Cleaning and Advanced Catalytic Emission Reduction Technology/College of Chemical Engineering, Beijing Institute of Petrochemical Technology, Beijing 102617, China 2 National Key Lab of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China

Abstract: The macroporous microspheres were prepared through suspension polymerization based on a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate. The effect of porogen on the microspheres structure was evaluated in terms of pore size and surface area. Porogen contained dichloromethane (δ = 9.7 (cal cm‒3)1/2) and N-octanol (δ = 10.3 (cal cm‒3)1/2) which were correspond to a good and poor solvent, respectively. The solubility parameter of porogen was controlled in the range of 9.89–10.09 (cal cm‒3)1/2. The pore size of microspheres increased with the increase of difference value of solubility parameter between the polymer and the porogen. On the contrary, the surface area of microspheres decreased. The anion exchange media was prepared through coupling poly(ethylene imine) in the microspheres and the proteins transport was determined by frontal analysis method. The macroporous microspheres with 257 nm pore size could still afford a high proteins capacity (45.1 mg mL‒1). These macroporous supports showed great potential in rapid separation of proteins. Key Words:

1

Porogen; Solubility parameter; Macroporous microspheres; Protein; Separation

Introduction

The high through-put separation of biomolecular has been a hot topic with the development of pharmaceuticals. Chromatography, as a main technology, plays an important role in separation and purification of proteins, in which the chromatographic support is the key of the technique. The supports based on the agarose are widely used for separation of proteins due to its good biocompatibility and chemical stability. However, this kind of matrix has poor mechanical strength, resulting in enduring the low operation pressure. The small pores of this support, which are in the range from 3 to 50 nm[1], bring a low binding capacity and throughput of proteins, especially for large biomolecules or particles, such as virus and virus-like particles. The macroporous supports based on the polymers show some advantages in terms of

high mechanical strength and large pore size (more than 100 nm). This kind of supports can tear more than ten megapascals and afford a rapid transport of large molecules such as proteins[2]. The agarose-based macroporous polymer supports are mainly prepared by suspension radical polymerization. In this method, it is not easily to fabricate the large through-put pore in the polymer microspheres. Hahn et al[3] reported a perfusion support that could allow protein transfer in convective way. POROS is the typical representatives of the perfusion chromatography that is composed of two kinds of pores, including throughpores (500–800 nm) and diffusion pores (20–100 nm). The pore structure was not easy to accurately control due to its complicated preparation process. The fabrication of pores needs two steps, production of nanoparticles and formation of aggregation[4]. Sun’s group[5,6]

________________________ Received 7 October 2017; accepted 21 November 2017 *Corresponding author. E-mail: [email protected] This work was supported by the Beijing Natural Science Foundation of China (No. 2162013), the Beijing Municipal Education Commission Science and Technology Project of China (No. KM201710017003), and the 2017BIPT-SPBYSJ of China (No. 17032021006). Copyright © 2018, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(17)61069-7

LAN Meng-Fei et al. / Chinese Journal of Analytical Chemistry, 2018, 46(2): 288–292

prepared gigaporous microsphere based on polymethacrylate, through compound porogen containing calcium carbonate particles and organic solvents. In this method, the throughpores were not well-distributed in the microspheres because of the poor compatibility between inorganic particles and organic solvents. Zhou et al[7,8] used reverse micelles swelling method to prepare a gigaporous supports. Design of porogen was proved to be an efficiency method for fabrication of the throughpores in the microspheres. These large pores in supports could improve the transport of proteins. Meanwhile, it was found that the supports with the large pores afforded low surface area. As a result, the binding capacity of proteins was low on these supports[9, 10]. It was concerned by many researchers that how to increase the capacity of proteins on the macroporous media. Our group ever used a method of atom transfer radical polymerization to prepare the polymethacrylate microspheres with the throughpores that were in the range from 200 to 300 nm[11,12]. In addition, the microspheres could contain higher surface area than ones from another ordinary polymerization method. Meanwhile, it was found that porogen had drastic influence on structure of the microspheres. But the detailed influences were not explored in previous studies. In present study, the effect of porogen on the microspheres structure was evaluated in terms of pore size, surface area, morphology, and binding capacity. The solubility parameter of porogen affecting the microspheres structure was also investigated.

2 2.1

Experimental Instrumentation and materials

Glycidyl methacrylate (GMA), ethylene glycol dimethacrylate (EDMA), poly(vinylalcohol) (PVA, Mw = 1700 ± 50, 87% alcoholysis degree), and poly(ethylene imine) (PEI, Mw = 600) were purchased from Aladdin Reagent Co., Ltd (Shanghai, China). Azobisisobutyronitrile (AIBN), dichloromethane (DCM), n-octanol (OA), and sodium lauryl sulfate (SDS) were produced by Shanghai Chemical Plant (Shanghai, China). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All the above chemicals were used as received. Pore diameter and surface are of the microspheres, in their dry state, were measured on an AutoPore IV 9500 mercury intrusion porosimetry (Micromeritics, USA) and specific surface area measuring instrument (V-Sorb 2800, Nanjing Gold APP Instruments, China). The morphologies of these microspheres were observed by scanning electron microscope (SEM) (FEI Quanta400F, FEI Lim. Co., Japan). The dynamic binding capacity of proteins was measured by ÄKTA Purifier 10 (GE, USA). 2.2

Methods

2.2.1

Preparation of macroporous microspheres by suspension polymerization

In a typical suspension polymerization, as shown in Fig.1, AIBN (0.040 g), GMA (1.0 mL), EDMA (1.0 mL), DCM (1.5 mL) and OA (1.5 mL) were added to an ampule. Then the reaction mixture was added into 50 mL of aqueous solution that contained 0.5 g of PVA and 0.05 g of SDS under stirring at 200 rpm. The reactor was heated to 60 ºC and the polymerization proceeded at 60 ºC for 8 h. The reaction solution was filtered and the resulted microspheres were then soxhlet extracted by acetone for 24 h. Finally, the microspheres were vacuum dried at 50 ºC for 24 h. After that, the dried microspheres were stored at room temperature. 2.2.2

Modification of PGMA-EDMA microspheres with PEI and determination of ion exchange capacity

To measure protein capacity of PGMA-EDMA microspheres, PEI was used to modify the supports to obtain anion exchange media. This derivatization was carried out according to previous study[13]. The mixture containing PGMA-EDMA (5.0 g), dimethyl sulfoxide (50 mL) and PEI (5.0 g) was heated to 60 ºC for 24 h. After the reaction finished, the microspheres (PGMA-EDMA-PEI) were filtered and washed with water. The ion exchange capacity of these microspheres was measured according to the previous method[14]. The detailed procedure was as the following steps: (1) 30 mL of NaOH (1.0 M) was passed through the column filled with the supports; (2) the column was cleaned with water to remove ‒OH adsorbing on the surface; (3) 50 mL of HCl (0.10 M) was passed through the column and collected in a flask; (4) 30 mL of NaCl (1.0 M) was passed through the column and collected in the same flask as described in step 3; (5) all of the collection was titrated by NaOH solution. The ion exchange capacity (Q) was calculated as follows: (1) C1V1 − C2V2 Q=

V0

where, C1 and C2 are the concentration of HCl and NaOH, V0, V1 and V3 are the volume of supports, HCl and NaOH, solutions respectively. 2.2.3

Determination of binding capacity of proteins by frontal analysis

Frontal breakthrough curves were measured for feeds

Fig.1 Scheme for preparation of PGMA-EDMA microspheres

LAN Meng-Fei et al. / Chinese Journal of Analytical Chemistry, 2018, 46(2): 288–292

consisting of 1.0 mg mL–1 bovine serum albumin (BSA) in Tris- HCl buffer solution (50 mM, pH 8.0) that was used as equilibration buffer. The column（Φ 10 × 13 mm）was packed with 1.0 mL of PGMA-EDMA-PEI. The adsorbate was detected using UV detector at 280 nm. The experiments were conducted on an AKTA Purifier 10. The first step was to equilibrate the column with 10 column volume (CVs) of buffer. Then the column was loaded with feed until 100% breakthrough was achieved. Excess feed was washed out of the column with equilibration buffer. The adsorbate was eluted using eluent (1.0 M NaCl in equilibration buffer). After re-equilibrating the column with buffer, the column was ready for the next run. Dynamic capacities were calculated according to the breakthrough curves.

amount of DCM decreased (DCM/OA, 1:1, V/V), pores less than 100 nm were observed in the surface of the microspheres (Fig.2, B1 and B2). Further decrease of DCM ratio (DCM/OA, 1:1.5, V/V) led to large pores (200–300 nm, Fig.2, C1 and C2). In the case of DCM/OA ratio of 1:2, the amount of large pores and pore size increased obviously in the microspheres. Meanwhile, it was found that the permeability of the supports was improved by many interconnected through pores. However, too little content of DCM (DCM/OA ratio of 1:3) in the porogen would make the microspheres to be broken. The above results indicated that OA, as a poor solvent, was in favor of large pore formation. On the contrary, DCM could improve the mechanical strength of the microspheres. These rules were similar with the ordinary porous materials [16].

3

3.2

3.1

Results and discussion Effect of porogen composition on the morphology of microspheres

The control of porogen composition plays an important role in the formation of pores in the supports. In this study, the effect of porogen composition on structure was examined using compound porogen consisting of dichloromethane (DCM) and n-octanol (OA). In which, DCM (δ = 9.7 (cal cm‒3)1/2) was a good solvent for polymer and OA (δ = 10.3 (cal cm‒3)1/2) was a poor solvent. The solubility parameter of PGMA-EDMA was 9.3 (cal cm‒3)1/2 according to the reported value[15]. The morphology of PGMA-EDMA showed an obvious change with the composition of porogen. The results are shown in Fig.2. The microspheres with DCM/OA ratio of 2:1 had a smooth surface without pores (Fig.2, A1 and A2). When the

Effect of solubility parameter on microsphere structure

The surface areas and pore sizes of microspheres with different ratios of porogen were summarized in Table 1. The solubility parameter drastically influenced the structure, such as surface area and pore size. The pore size increased with the value of solubility parameters. On the contrary, the surface area had a decrease trend. It was found that, when the solubility parameter of solvent is close to that of polymer, it can well dissolve this polymer[17]. Therefore, the difference between the solubility parameters of microspheres and porogen can be considered to be an indicator of compatibility of microspheres and solvent, which may in turn affect the structure of microspheres as shown in Fig.3 and Fig.4. The pore size of microspheres raised with the increase of ∆δ. But the surface area became smaller and smaller with the increase

Fig.2 Morphology of PGMA-EDMA microspheres by porogen with different ratios of DCM and OA: A(1, 2), B(1, 2), C(1, 2) and D(1, 2) are corresponding to ratio of 2:1, 1:1, 1:1.5, 1:2, respectively Table 1 Surface area and pore size of PGMA-EDMA obtained by porogen with different ratio of DCM and OA Parameter Pore size* (nm) Surface area** (m2 g-1) Solubility parameter ((cal cm‒3)1/2) *

DCM/OA (V/V) 2:1

1:1

1:1.5

1:2

21 ± 0.2 134 ± 1 9.89

73 ± 0.2 91 ± 1 10.0

124 ± 0.2 67 ± 1 10.06

257 ± 0.2 47 ± 1 10.09

The average pore size is determined by mercury intrusion method; ** The surface area is measured by nitrogen adsorption method.

LAN Meng-Fei et al. / Chinese Journal of Analytical Chemistry, 2018, 46(2): 288–292

3.3

Fig.3 Relationship of ∆δ and pore size of microspheres

The derived microspheres, PGMA-EDMA-PEI, were evaluated in terms of its ion exchange capacity and dynamic binding capacity, and the results are shown in Table 2. The ion exchange capacities showed a slight decrease with the increase of pore sizes. The possible reason was that the amount of PEI coupled onto the microspheres decreased with the decrease of surface area. But this had no obvious influence on the ion exchange capacity which remained more than 0.35 M for all tested microspheres. The dynamic binding capacities were expected to decrease with the decrease of surface area. As shown Table 2, the dynamic binding capacity indeed decreased from 61.4 mg mL–1 to 45.1 mg mL–1. The corresponding reduction rate of 26.5% was far less than the decline rate of 35.1% of the surface area. These results indicated that this microporous supports had a high capacity for proteins.

4

Fig.4 Relationship of ∆δ and surface area of microspheres

of ∆δ. As described above, the poor compatibility between PGMA-EDMA and porogen, corresponding to large ∆δ, could cause early phase separation in the polymerization. The earlier phase separation occurred, the larger aggregation of the cluster would be obtained. Thus, the pore size from the gaps between the aggregations increased while the surface area decreased due to large clusters forming on the microspheres.

Dynamic binding capacity of proteins on microspheres with different structures

Conclusions

The microporous microspheres, based on a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, were prepared by suspension polymerization. The results showed that the solubility parameter affected the microspheres structure. The pore size increased and the surface area decreased with the increase ratio of poor solvent in the porogen. The less different value of solubility parameter would result in the smaller pore size, while the larger surface area and higher capacity for proteins could be obtained in the microspheres. These results provided a reference basis for designing the chromatographic media.

Table 2 Ion exchange capacity and dynamic binding capacity of PGMA-EDMA-PEI with different structure Parameter Pore size (nm) Surface area (m2 g‒1) Ion exchange capacity (M) Dynamic binding capacity* ( mg mL‒1)*

DCM/OA (V/V) 2:1 21 ± 0.2 134 ± 1 0.398 ± 0.002 61.4 ± 0.1

1:1 73 ± 0.2 91 ± 1 0.373 ± 0.002 50.2 ± 0.1

1:1.5 124 ± 0.2 67 ± 1 0.370 ± 0.002 48.2 ± 0.1

1:2 257 ± 0.2 47 ± 1 0.355 ± 0.002 45.1 ± 0.1

*

The dynamic binding capacity was obtained by 50% breakthrough.

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