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Covalently coating dextran on macroporous polyglycidyl methacrylate microsphere enabled rapid protein chromatographic separation
Materials Science and Engineering C 32 (2012) 2628–2633
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Covalently coating dextran on macroporous polyglycidyl methacrylate microsphere enabled rapid protein chromatographic separation Rongyue Zhang, Qiang Li, Juan Li, Weiqing Zhou, Peili Ye, Yang Gao, Guanghui Ma ⁎, Zhiguo Su National Key Lab of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
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Article history: Received 29 March 2012 Received in revised form 19 June 2012 Accepted 10 August 2012 Available online 20 August 2012 Keywords: Materials Hydrophilization Macroporous microsphere Rapid protein separation Covalently coating
a b s t r a c t Protein denaturation and nonspecific adsorption on polymer media as a chromatographic support have been a problem which needs to be overcome. Macroporous poly(glycidyl methacrylate–divinylbezene) (PGMA– DVB) microspheres prepared in this study were firstly covalently coated with dextran through a three-step method. The dextran was firstly adsorbed onto the microspheres and then covalently bound to the PGMA– DVB microsphere through ether bonds which were formed by hydroxyl group reacting with epoxy group at the presence of 4-(Dimethylamino) pyridine. Finally, the coating dextran layer was crosslinked by ethylene glycol diglycidyl ether to form the continuous network coating. The coated microspheres were characterized by Fourier transform infrared spectra, scanning electron microscope, mercury porosimetry measurements, laser scanning confocal microscope, and protein adsorption experiments. Results showed that PGMA–DVB microspheres coated with dextran successfully maintained the macroporous structure and high permeability. The backpressure was only 1.69 MPa at a high flow rate of 2891 cm/h. Consequently, the hydrophilicity and biocompatibility of modified microspheres were greatly improved, and the contact angle decreased from 184° to 13°, and nonspecific adsorption of proteins was decreased to little or none. The clad dextran coating with large amounts of hydroxyl group was easily derived to be various functional groups. The derived media have great potential applications in rapid protein chromatography. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Microspheres based on polymer matrix, as chromatographic separation media, have been popular in separation field [1–4]. Especially, the interest in and demand for high throughput separation of biomolecules accelerated the exploitation of effective polymer bioseparation media in chromatography. The usual polymer media were polystyrene or polyacrylate matrix due to their high chemical and mechanical stability at a wide pH range (pH = 0–14),such as POROS™ and TOYOPEARLS™. However, to use this packed material for the chromatography of proteins, it is essential to enable the matrix surface to be more hydrophilic in order to avoid irreversible interactions or protein denaturation. The alternative method is surface hydrophilization of the hydrophobic polymers by using hydrophilic polymer. Hydrophilization of polystyrene-divinylbenzene (PS-DVB) particles was extensively studied through adsorption of nonionic polymers such as ethylhydroxyethyl cellulose [5,6], poly(ethylene oxide)–poly(propylene oxide) copolymers [7,8], polyglycerol [9], or dextran [10]. However, these polymers partially desorbed when exposed to other polymers or proteins [11,12]. To overcome this problem, in a previous study, we introduced low concentrations of hydrophobic group onto the agarose which was used to coat the PS-DVB microsphere [13]. The coating agarose was relatively stable, ⁎ Corresponding author. Tel./fax: +86 10 82627072. E-mail address: [email protected] (G. Ma). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.08.021
and could be tolerant to 1 M NaOH or 1 M HCl at room temperature. However, all the methods described above showed that molecules of solute comprising hydrophilic and hydrophobic domains were adsorbed onto the hydrophobic polymeric support. The molecules of coating were chemically crosslinked with each other to form a continuous layer, and the crosslinked layer was adhered to the polymeric matrix surface by hydrophobic–hydrophobic interaction not by chemical bond. This physically adsorptive process was not stable enough, and was easily destroyed in some derivation reaction conditions, such as strong acidic or basic conditions, or heated condition. In order to obtain more stable hydrophilic layer on the polymeric support surface, an effective method was that the hydrophilic coating was covalently bound onto the hydrophobic surface. We ever performed a two-step reaction to coat PS-DVB microspheres using polyvinyl alcohol (PVA), including chloroacetylation of PS-DVB microspheres and covalently bonding PVA on microspheres by Williamson reaction afterward [14]. The hydrophilicity of modified PS-DVB microspheres was significantly improved, but the nonspecific adsorption of protein was still more than 5.0 mg/g dry microspheres due to the hydrogen bond formed by the hydroxyl groups of PVA and carboxyl group of proteins. To further decrease the nonspecific adsorption of proteins on the microspheres, the dextran was attempted to be used as hydrophilic coating for hydrophilization of microspheres in this study. Our research group has developed an easy and novel method to prepare macroporous poly(glycidyl methacrylate-divinylbezene) (PGMA–
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DVB) microspheres with pore size of about 200–500 nm [15], which were used as a perfusion chromatographic support [16–18]. This macroporous PGMA–DVB microsphere, as modified support, has its distinctive advantages. One is that its macropores (more than 200 nm) facilitate hydrophilic polymer diffusion into the interior of the microsphere compared to the conventional porous polymer particles with 10–30 nm pores. Another is that PGMA–DVB surface has high content of epoxy groups, which easily react with hydroxyl groups of the hydrophilic molecules [19,20]. Meanwhile, the surface of PGMA–DVB shows weak hydrophobicity due to lots of epoxy group compared with those of PS-DVB. So, the PGMA–DVB microsphere adsorbs hydrophilic molecules easier than PS-DVB does. Therefore, hydrophilization of PGMA– DVB has been expected to obtain better hydrophilic separation media. In this study, the macroporous PGMA–DVB microspheres was hydrolyzed through chemically coating dextran on the surface of microsphere for the first time. Dextran, a biocompatible natural polysaccharide and a good coating layer, cannot only effectively shield the PGMA–DVB surface from hydrophobic interaction, but also provides a neutral and easily derivative layer [21,22]. In this study, dextran can be covalently bound onto the PGMA–DVB microspheres through ester bond formed by reaction between hydroxyl groups of dextran and epoxy groups of PGMA–DVB [23]. In order to shield the hydrophobic surface of PGMA–DVB, this covalently coated dextran was crosslinked by some crosslinkers and formed a network continuous coat. The coated microspheres were characterized by various testing methods and showed high hydrophilicity and stability. Further, the coated microsphere could maintain the macropore structure and ensure high throughput for perfusion chromatography. Therefore, the coated microspheres are a potential media for rapid protein chromatography.
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microspheres were dried under vacuum at 60 °C until constant weight for the covalently bonding dextran ratio (CBDR) calculation according to Formula 1b. In order to verify whether the epoxy groups of microsphere hydrolyze in the presence of DMAP, the control experiment without addition of dextran was carried out and the epoxy value of the resulting microspheres was determined by the hydrochloric acid–acetone method [24]. The third step was that the covalently coated layer of dextran was crosslinked with ethylene glycol diglycidyl ether (EDGE) at various concentrations in 5 M NaOH (20 mL/g dry PGMA–DVB microspheres) at 25 °C for 24 h. Finally, the resulting particles were soxhlet extracted by water to remove the residual unreacted compounds for 48 h. The final microspheres were dried under vacuum at 60 °C until constant in weight and were characterized by FTIR (JASCO FT/IR-400/600). The dried microspheres (0.5%) were mixed with potassium bromide (KBr) powder and ground to homogeneous find powder. Then the mixture was added into the mold for preparation of pellet. The pellet was inserted into the IR sample holder and the spectrum was obtained. Formula 1 : a…CW ¼
b…CBDR ¼
W 2 −W 1 W1
W 3 −W 1 100% W 2 −W 1
In Formula 1, W1 and W2 are the dry PGMA–DVB microsphere weight before and after adsorption of dextran, respectively, and W3 represents the weight of the dry PGMA–DVB microspheres after covalently bonding dextran. 2.3. Evaluations of surface hydrophilicity
2. Experimental section 2.1. Materials The macroporous poly(glycidyl methacrylate-divinylbezene) (PGMA–DVB) microspheres used in this study were synthesized by a modified suspension polymerization process developed in our previous study [15]. The specific surface area was 54 m2/g, the average diameter was 55 μm (30–85 μm range), and the average pore size was 230 nm (100–500 nm range). The microspheres were extracted with acetone in a Soxhlet extractor for 24 h and then dried under vacuum at 50 °C. Dextran powder was purchased from Seebio Corp. Bovine serum albumin (BSA), ovalbumin and 4-(Dimethylamino) pyridine (DMAP) were obtained from Sigma-Aldrich (USA). Ethylene glycol diglycidyl ether (EDGE; AR) was from Sinopharm Chemical Reagent Co., Ltd. All reagents were used as received. 2.2. Covalently coating macroporous PGMA–DVB microspheres with dextran The covalent coating procedure was carried out by three steps. The first step was physical adsorption of dextran onto the PGMA–DVB microsphere; the specific step was that the microspheres (1.0 g) and dextran (1.0 g) in DMSO solution (20 mL solution/g dry PGMA– DVB microspheres) were stirred at 25 °C for 24 h; after that the suspension was filtered through a sintered glass funnel and washed with DMSO (200 mL/g of dry PGMA–DVB microspheres); at last, the washed microspheres adsorbed with dextran were dried under vacuum at 60 °C until a constant weight for the adsorption quality calculation according to Formula 1a. The second step was as follows: the PGMA–DVB microspheres adsorbed with dextran were transferred to the three-necked flask containing 20 mL acetone, 0.16 g DMAP and this chemical bonding reaction was kept for 72 h at 37 °C; then the covalently coated PGMA–DVB microspheres were soxhlet extracted by water to remove the residual unreacted compounds for 48 h; after that the coated
An optical contact angle measuring device (OCA20, Dataphysics, Germany) was employed to study the surface hydrophilicity of PGMA– DVB microspheres before and after coating. The contact angle of microspheres was measured by using the pressed-disk technique. The microspheres were firstly pressed into a disk by a mold, and then the water was dropped onto the disk by a needle at a speed of 0.5 μL/drop. 2.4. Determination of BSA and ovalbumin irreversible adsorption on microspheres The hydrophobic adsorption of protein solutions (5 mg/mL) of bovine serum albumin (BSA) and ovalbumin was tested on the microspheres. The PGMA–DVB microspheres coated with dextran were filled into φ4.6 × 50 mm stainless steel column by slurry packing method on a SHIMADZU LC-8A (Japan). The mobile phase was 50 mM phosphate buffer (pH 7.0 at 1 mL/min, with an injection volume of 200 μL). Repeated 5 injections of proteins were carried out on the SHIMADZU LC-8A (Japan). After each injection, the column was washed with 10 column volumes (CV) of 1.0 M NaOH for the column clean process [25,26]. All chromatography processes were performed at ambient temperature. Repeated injections of a protein should result in a constant peak area if there is no irreversible adsorption of proteins. The uncoated PGMA–DVB microsphere was also evaluated as a control batch. Super Fluor 488-labeling BSA (SF488-BSA) adsorption experiments were also conducted in a 4 °C incubator, after 24 h adsorption, the microspheres were separated from the SF488-BSA solution by centrifugation and washed with PBS 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 a TCS SP2 laser scanning confocal microscope (LCSM) (Leica, Germany) to visualize the adsorption of SF488-BSA onto microspheres. The samples were detected at 488 nm, and the fluorescent images at 520– 550 nm wavelengths were then taken.
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2.5. Measurement of microspheres pore diameter and morphology Pore diameter measurement of PGMA–DVB before and after coating was conducted by an AutoPore IV 9500 mercury porosimetry (Micromeritics, USA) to study the difference between PGMA–DVB microspheres and PGMA–DVB–dextran. Experiments were performed in accordance with the protocol given in the AutoPore IV 9500 operator's manual. The morphologies of the microsphere before and after coating were observed by scanning electron micrograph (SEM) (JSM-6400, JEOL Lim. Co., Japan). The detailed method of the sample preparations for SEM observation was described as follows. Microspheres were re-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 was coated with a thin gold film under reduced pressure below 5 Pa with a JFC-1600 fine coater (JEOL, Japan). 2.6. Mechanical stability and permeability of media The PGMA–DVB–dextran microspheres were packed into aφ4.6 × 50 mm stainless steel column as described on Chapter 2.4. The mechanical stability and permeability of the media were evaluated through measuring the backpressures of column at different flow rates, and the mobile phase was 50 mM phosphate buffer (pH 7.0).
transesterification and resulted in the direct attachment of methacryloyl groups at the dextran [23] (Scheme 1). On the basis of basicity, the order of reactivity was OH-2>OH-3> OH-4 of the glucopyranose ring [28]. However, steric hindrance at position 2 might induce substitution of OH-3. Van Dijk-Wolthuis studied the reaction of GMA with dextran in DMSO-d6 via NMR spectra and found that the substitution positions were at OH-2 and OH-3 in a 1:1 ratio [23]. Thus, the hydrophilic coating was covalently bound to the PGMA–DVB microspheres. Fig. 2 showed that the covalently bonding dextran ratio (CBDR) increased firstly then decreased at the titer range of 0.032–0.26 mol/L DMAP. DMAP, as catalyst, could accelerate the epoxy group to react with hydroxyl group of the dextran [29]. Therefore, the covalently bonding dextran ratio (CBDR) increased with increase of DMAP in the early stage. Why not increase but decrease with the rise of DMAP in a later stage? The possible cause was that DMAP was adsorbed on the PGMA–DVB microsphere surface which occupied a number of adsorption sites in the microspheres resulting in desorption of the pre-adsorbed dextran on the microspheres. Excessive DMAP exacerbated desorption of dextran from the surface of microspheres. In our previous study [13], the similar phenomenon found that the addition of excessive NaOH led to desorption of the modified agarose from the surface of PS-DVB microspheres. So, it was found that CBDR decreased with the increase of DMAP in the latter. To ensure enough dextran to be covalently bonding on the microsphere, the DMAP amounts were chosen to be 0.13 mol/L.
3. Results and discussion 3.1. Physical adsorption of dextran on macroporous PGMA–DVB microspheres The adsorption amounts of dextran with various molecule weights on PGMA–DVB microspheres were shown in Fig. 1. The adsorption quality could reach the maximum in 24 h. The adsorption quality decreased with the increase of molecule weights of the coated dextran. Dextran of larger molecule weight has bigger size than those of low molecular weights [27], so that dextran with larger molecule weight was more difficult to diffuse into the inner of PGMA–DVB microspheres than those of low molecular weights. To clothe well the hydrophobic surface, the dextran with 5 K molecular weight and 24 h-adsorption time was selected for the further coating study. 3.2. Effect of DMAP amounts on covalently bonding dextran ratio on PGMA–DVB microspheres DMAP was a basic catalyst for opening the epoxy groups to react with the adsorbed dextran. In the present of DMAP, the epoxy groups in the pore surface reacted with the hydroxyl groups of dextran via
Fig. 1. Adsorption quality of dextran with various molecular weights on PGMA–DVB microspheres. Adsorption condition: dextran/microspheres/DMSO = 1:1:20 (g/g/mL), mechanical stirring for different times at room temperature.
3.3. Effect of reaction time and temperature on covalently bonding dextran ratio on PGMA–DVB microspheres Fig. 3 showed that CBDR increased first then tended to a stable value with the reaction time. In the early stage, the amounts of epoxy groups in the PGMA–DVB were higher, which were consumed by the reaction of dextran. Therefore, the covalently coated dextran reaction rates were high and its amounts increased along with the reaction time. As the reaction was carried on, the epoxy group amounts decreased to a relatively low value, thus, little dextran could react with PGMA–DVB microspheres. Therefore, the covalently coated dextran amounts turned to be the constant value in the final reaction time. From Fig. 3, it was found that the maximum amounts of covalently coated dextran had been obtained when the reaction time reached 60 h. Fig. 4 showed the effect of temperature. CBDR showed first increase and then decrease tendency at the temperature range of 17–57 °C. Increased temperature accelerated reactive rate of dextran and microsphere [29] and CBDR increased with temperature in the early stage. However, CBDR began to decrease when temperature reached 37 °C. The possible reason was higher temperature accelerates desorption of dextran on PGMA–DVB. Generally, high temperature should not be favor increasing the adsorption quantity which resulted from higher diffusion rate caused by the elevated temperature [30–32]. In order to obtain high coating amount, the reaction temperature needed to be controlled in this reaction. From Fig. 4, the covalently coated dextran reaction rate reach maximum
Scheme 1. PGMA–DVB microspheres react with dextran at the existence of DMAP.
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Fig. 2. Effect of DMAP concentrations on the covalently bonding dextran ratio.
at 37 °C. Therefore, the reaction temperature was selected to be 37 °C.
3.4. Effect of EDGE on the hydrophilicity of covalently coated PGMA–DVB microspheres with dextran Table 1 showed effect of EDGE. The contact angle of the uncoated PGMA–DVB microsphere was 134 ° (Sample 1 #). After covalently coating, it dropped to be 78 ° (Sample 2 #). Although the hydrophilicity of the microspheres had been improved to some extent after coating dextran, this value (78°) indicated that the coating dextran could not fully shield the hydrophobic surface of the PGMA–DVB microspheres. We ever found that agarose could form a unique coating layer after crosslinkage by EDGE [13]. EDGE was also extensively applied to crosslinking many biomaterials [33,34], which had good biocompatibility. In this study, EDGE was attempted to crosslink the coating dextran for forming a uniform coat on the microsphere surface. The contact angle decreased with increase of EDGE and the lowest value dropped to 11° when the concentration of EDGE reached 0.046 mol/ L. Unfortunately, the coated microspheres were aggregated at the 0.046 mol/L EDGE. Therefore, the amounts of EDGE were controlled to be less than 0.046 mol/g dry microspheres. The coated microspheres were characterized by FTIR and shown in Fig. 5. Comparing these three microspheres, the intensity of the peak at 3457 cm −1, which was attributed to –O–H stretching vibration of hydroxyl groups, was increased as the following order: Fig. 5ab Fig. 5bb Fig. 5c (Fig. 5a: Sample 1#; Fig. 5b: Sample 2#; Fig. 5c: Sample 7#). Additionally, the intensity of the peak at 1102 cm−1, which was attributed to C–O–C stretching vibration of ester bond, was also increased according to the above order. These indicated that the dextran had been successfully covalently coated onto the PGMA–DVB microspheres.
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Fig. 4. Effect of reaction time on covalently bonding dextran ratio.
Meanwhile, it also confirmed that the coated dextran had been efficaciously crosslinked by EDGE.
3.5. Pore diameter and morphology of PGMA–DVB microspheres before and after covalently coated dextran The pore diameters of microspheres (Sample 1 # and Sample 7 #) were measured by mercury porosimetry measurements and shown in Fig. 6. The average pore diameters were slightly changed from 221 nm to 201 nm after coating. In our previous study, the modified agarose (Mw = 24,000) was used to physically coat PS-DVB microspheres and had no influence on macroporous structure of microspheres [35]. In this study, PGMA–DVB coated with dextran (Mw = 5000) could also maintain the macropores and ensure high permeability. Additionally, the morphology of PGMA-DVB microsphere before (Sample 1 #) and after covalently coated (Sample 7 #) was also observed by SEM. In Fig. 7, coating dextran was observed on the surface of coated microspheres (Fig. 7B1) compared to the smooth surface of uncoated microspheres (Fig. 7A1). Meanwhile, the macropores of microspheres after coating were still preserved and not clogged by coating dextran (Fig. 7B and B1, Sample 7 #). The macropores of PGMA–DVB microspheres were a key feature for using perfusion chromatography because the convective mass transfer was realized through macropores in the media [36]. In order to verify whether the coating layer affected the permeability of the microspheres, the backpressures were measured with different flow rates on the microspheres (Sample 1 # and Sample 7 #) and shown in Fig. 8. The backpressure of the coated PGAM–DVB with dextran (Sample 7 #) was at the range of 0.3–1.7 MPa when the flow rate changed from 1 mL/min to 8 mL/min. The back pressure of Sample 7 # was slightly increased compared to the uncoated PGMA–DVB microsphere. In the meantime, the back pressure could keep a linear
Table 1 Contact angles of different PGMA–DVB microspheres. Sample no.
Fig. 3. Effect of reaction time on covalently bonding dextran ratio.
1# 2# 3# 4# 5# 6# 7# 8#
Covalently coated dextran (mg/g dry microsphere)
0 65 65 65 65 65 65 65
EDGE
Contact angle
(mol/ L)
(°)
0 0 0.013 0.019 0.026 0.033 0.039 0.046
134 78 65 45 32 17 13 11
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Fig. 5. FTIR of PGMA–DVB microspheres, a Sample 1#; b Sample 2#; c Sample 7#.
increase as the flow rate increased in Fig. 8. This indicated that the macropore structure and mechanical strength were stable even at high flow rate [37]. From all the above tests, it can be concluded that the coated dextran could reserve the macropores and has no influence on the application of the coated microspheres in perfusion chromatography. 3.6. Evaluation of BSA and ovalbumin irreversible adsorption on microsphere The adsorption of BSA and ovalbumin on the microspheres was shown in Fig. 9. The protein mass amounts were 0.40 mg for each injection and the chromatography peak areas of the repeated 5 injections were constant in the coated PGMA–DVB microspheres (Sample 7 #). These constant peak areas confirmed almost no adsorption of proteins on Sample 7 # [38]. Also, it indicated that the coated dextran layer was stable even after repeated washing with strong basic solution (1.0 M NaOH). However, the peak areas on uncoated PGMA– DVB microspheres (Sample 1 #) were less ten times than those on Sample 7 #. Moreover, the inconstant peak areas increased with the rise of the injection numbers. These indicated that the tested proteins had a large number of irreversible adsorption onto the uncoated microsphere (Sample 1 #). It was improved that the coated dextran could clothe well the hydrophobic surface of PGMA–DVB microspheres. Additionally, the coated microspheres (Sample 7 #) were immersed into 1.0 M HCl solution for 12 h and tested its stability in acid environment. The immersed microspheres were tested using the above-mentioned BSA and ovalbumin adsorption testing and almost no adsorption of proteins on it. This indicated that the coated microspheres were stable in the pH range of 0–14. In order to observe the hydrophilicity of PGMA–DVB microsphere surfaces more straightly, the SF488-BSA adsorption experiment was carried out on Sample
Fig. 6. Pore diameter distribution of microspheres, ■Sample 7#; ●Sample 1#.
Fig. 7. SEM of PGMA–DVB microsphere, A and A1 is Sample 1#; B and B1 is Sample 7#.
1 # and Sample 7 #. As shown in Fig. 10, great fluorescent BSA was observed on Sample 1 # (Fig. 10A), while little fluorescence was detected in Sample 7 # (Fig. 10B). This comparative study provided a straight insight on increasing hydrophilicity of PGMA–DVB microsphere coated with dextran.
4. Conclusions A potential hydrophilic packing support has been developed by covalently coating macropores of PGMA–DVB microspheres with dextran. The hydrophilicity of PGMA–DVB after coated with dextran was significantly increased when the coating dextran layer was crosslinked by EDGE. The optimized EDGE concentrations were 0.039 M. After coating, the hydrophilicity and biocompatibility of the PGMA–DVB microspheres were greatly enhanced. The nonspecific adsorption of proteins was nearly reduced to none and the coating dextran layer was stable after washing with 1 M NaOH solution. The macropore structure and morphology of the microspheres were not changed after coating, and high throughput could be realized on this coated media due to its high permeability. This study provided a hydrophilic chromatographic support for different types of chromatography such as ion-exchange or affinity chromatography. After further derivation, the coated microspheres would be a prospective stationary phase in high-speed preparative protein chromatography. The further application for protein separation will be submitted to a journal in future.
Fig. 8. The backpressures at various flow rates, red cycle, Sample 7#; black square, Sample 1#.
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Fig. 9. Graph of chromatography peak area versus injection number in BSA and ovalbumin adsorption by the microspheres, ● BSA on Sample 7#; ■ Ovalbumin on Sample 7#;▲ Ovalbumin on Sample 1#;▼ BSA on Sample 1#.
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Fig. 10. LCSM of SF488-BSA adsorption on microsphere, A, Sample 1#; B, Sample 7#.
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Acknowledgment We would like to thank the financial support from the National Natural Science Foundation of China (No. 51103158, No. 20906094 and KSCX2-EW-G-3).
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Covalently coating dextran on macroporous polyglycidyl methacrylate microsphere enabled rapid protein chromatographic separation Rongyue Zhang, Qiang Li, Juan Li, Weiqing Zhou, Peili Ye, Yang Gao, Guanghui Ma ⁎, Zhiguo Su National Key Lab of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
a r t i c l e
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Article history: Received 29 March 2012 Received in revised form 19 June 2012 Accepted 10 August 2012 Available online 20 August 2012 Keywords: Materials Hydrophilization Macroporous microsphere Rapid protein separation Covalently coating
a b s t r a c t Protein denaturation and nonspecific adsorption on polymer media as a chromatographic support have been a problem which needs to be overcome. Macroporous poly(glycidyl methacrylate–divinylbezene) (PGMA– DVB) microspheres prepared in this study were firstly covalently coated with dextran through a three-step method. The dextran was firstly adsorbed onto the microspheres and then covalently bound to the PGMA– DVB microsphere through ether bonds which were formed by hydroxyl group reacting with epoxy group at the presence of 4-(Dimethylamino) pyridine. Finally, the coating dextran layer was crosslinked by ethylene glycol diglycidyl ether to form the continuous network coating. The coated microspheres were characterized by Fourier transform infrared spectra, scanning electron microscope, mercury porosimetry measurements, laser scanning confocal microscope, and protein adsorption experiments. Results showed that PGMA–DVB microspheres coated with dextran successfully maintained the macroporous structure and high permeability. The backpressure was only 1.69 MPa at a high flow rate of 2891 cm/h. Consequently, the hydrophilicity and biocompatibility of modified microspheres were greatly improved, and the contact angle decreased from 184° to 13°, and nonspecific adsorption of proteins was decreased to little or none. The clad dextran coating with large amounts of hydroxyl group was easily derived to be various functional groups. The derived media have great potential applications in rapid protein chromatography. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Microspheres based on polymer matrix, as chromatographic separation media, have been popular in separation field [1–4]. Especially, the interest in and demand for high throughput separation of biomolecules accelerated the exploitation of effective polymer bioseparation media in chromatography. The usual polymer media were polystyrene or polyacrylate matrix due to their high chemical and mechanical stability at a wide pH range (pH = 0–14),such as POROS™ and TOYOPEARLS™. However, to use this packed material for the chromatography of proteins, it is essential to enable the matrix surface to be more hydrophilic in order to avoid irreversible interactions or protein denaturation. The alternative method is surface hydrophilization of the hydrophobic polymers by using hydrophilic polymer. Hydrophilization of polystyrene-divinylbenzene (PS-DVB) particles was extensively studied through adsorption of nonionic polymers such as ethylhydroxyethyl cellulose [5,6], poly(ethylene oxide)–poly(propylene oxide) copolymers [7,8], polyglycerol [9], or dextran [10]. However, these polymers partially desorbed when exposed to other polymers or proteins [11,12]. To overcome this problem, in a previous study, we introduced low concentrations of hydrophobic group onto the agarose which was used to coat the PS-DVB microsphere [13]. The coating agarose was relatively stable, ⁎ Corresponding author. Tel./fax: +86 10 82627072. E-mail address: [email protected] (G. Ma). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.08.021
and could be tolerant to 1 M NaOH or 1 M HCl at room temperature. However, all the methods described above showed that molecules of solute comprising hydrophilic and hydrophobic domains were adsorbed onto the hydrophobic polymeric support. The molecules of coating were chemically crosslinked with each other to form a continuous layer, and the crosslinked layer was adhered to the polymeric matrix surface by hydrophobic–hydrophobic interaction not by chemical bond. This physically adsorptive process was not stable enough, and was easily destroyed in some derivation reaction conditions, such as strong acidic or basic conditions, or heated condition. In order to obtain more stable hydrophilic layer on the polymeric support surface, an effective method was that the hydrophilic coating was covalently bound onto the hydrophobic surface. We ever performed a two-step reaction to coat PS-DVB microspheres using polyvinyl alcohol (PVA), including chloroacetylation of PS-DVB microspheres and covalently bonding PVA on microspheres by Williamson reaction afterward [14]. The hydrophilicity of modified PS-DVB microspheres was significantly improved, but the nonspecific adsorption of protein was still more than 5.0 mg/g dry microspheres due to the hydrogen bond formed by the hydroxyl groups of PVA and carboxyl group of proteins. To further decrease the nonspecific adsorption of proteins on the microspheres, the dextran was attempted to be used as hydrophilic coating for hydrophilization of microspheres in this study. Our research group has developed an easy and novel method to prepare macroporous poly(glycidyl methacrylate-divinylbezene) (PGMA–
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DVB) microspheres with pore size of about 200–500 nm [15], which were used as a perfusion chromatographic support [16–18]. This macroporous PGMA–DVB microsphere, as modified support, has its distinctive advantages. One is that its macropores (more than 200 nm) facilitate hydrophilic polymer diffusion into the interior of the microsphere compared to the conventional porous polymer particles with 10–30 nm pores. Another is that PGMA–DVB surface has high content of epoxy groups, which easily react with hydroxyl groups of the hydrophilic molecules [19,20]. Meanwhile, the surface of PGMA–DVB shows weak hydrophobicity due to lots of epoxy group compared with those of PS-DVB. So, the PGMA–DVB microsphere adsorbs hydrophilic molecules easier than PS-DVB does. Therefore, hydrophilization of PGMA– DVB has been expected to obtain better hydrophilic separation media. In this study, the macroporous PGMA–DVB microspheres was hydrolyzed through chemically coating dextran on the surface of microsphere for the first time. Dextran, a biocompatible natural polysaccharide and a good coating layer, cannot only effectively shield the PGMA–DVB surface from hydrophobic interaction, but also provides a neutral and easily derivative layer [21,22]. In this study, dextran can be covalently bound onto the PGMA–DVB microspheres through ester bond formed by reaction between hydroxyl groups of dextran and epoxy groups of PGMA–DVB [23]. In order to shield the hydrophobic surface of PGMA–DVB, this covalently coated dextran was crosslinked by some crosslinkers and formed a network continuous coat. The coated microspheres were characterized by various testing methods and showed high hydrophilicity and stability. Further, the coated microsphere could maintain the macropore structure and ensure high throughput for perfusion chromatography. Therefore, the coated microspheres are a potential media for rapid protein chromatography.
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microspheres were dried under vacuum at 60 °C until constant weight for the covalently bonding dextran ratio (CBDR) calculation according to Formula 1b. In order to verify whether the epoxy groups of microsphere hydrolyze in the presence of DMAP, the control experiment without addition of dextran was carried out and the epoxy value of the resulting microspheres was determined by the hydrochloric acid–acetone method [24]. The third step was that the covalently coated layer of dextran was crosslinked with ethylene glycol diglycidyl ether (EDGE) at various concentrations in 5 M NaOH (20 mL/g dry PGMA–DVB microspheres) at 25 °C for 24 h. Finally, the resulting particles were soxhlet extracted by water to remove the residual unreacted compounds for 48 h. The final microspheres were dried under vacuum at 60 °C until constant in weight and were characterized by FTIR (JASCO FT/IR-400/600). The dried microspheres (0.5%) were mixed with potassium bromide (KBr) powder and ground to homogeneous find powder. Then the mixture was added into the mold for preparation of pellet. The pellet was inserted into the IR sample holder and the spectrum was obtained. Formula 1 : a…CW ¼
b…CBDR ¼
W 2 −W 1 W1
W 3 −W 1 100% W 2 −W 1
In Formula 1, W1 and W2 are the dry PGMA–DVB microsphere weight before and after adsorption of dextran, respectively, and W3 represents the weight of the dry PGMA–DVB microspheres after covalently bonding dextran. 2.3. Evaluations of surface hydrophilicity
2. Experimental section 2.1. Materials The macroporous poly(glycidyl methacrylate-divinylbezene) (PGMA–DVB) microspheres used in this study were synthesized by a modified suspension polymerization process developed in our previous study [15]. The specific surface area was 54 m2/g, the average diameter was 55 μm (30–85 μm range), and the average pore size was 230 nm (100–500 nm range). The microspheres were extracted with acetone in a Soxhlet extractor for 24 h and then dried under vacuum at 50 °C. Dextran powder was purchased from Seebio Corp. Bovine serum albumin (BSA), ovalbumin and 4-(Dimethylamino) pyridine (DMAP) were obtained from Sigma-Aldrich (USA). Ethylene glycol diglycidyl ether (EDGE; AR) was from Sinopharm Chemical Reagent Co., Ltd. All reagents were used as received. 2.2. Covalently coating macroporous PGMA–DVB microspheres with dextran The covalent coating procedure was carried out by three steps. The first step was physical adsorption of dextran onto the PGMA–DVB microsphere; the specific step was that the microspheres (1.0 g) and dextran (1.0 g) in DMSO solution (20 mL solution/g dry PGMA– DVB microspheres) were stirred at 25 °C for 24 h; after that the suspension was filtered through a sintered glass funnel and washed with DMSO (200 mL/g of dry PGMA–DVB microspheres); at last, the washed microspheres adsorbed with dextran were dried under vacuum at 60 °C until a constant weight for the adsorption quality calculation according to Formula 1a. The second step was as follows: the PGMA–DVB microspheres adsorbed with dextran were transferred to the three-necked flask containing 20 mL acetone, 0.16 g DMAP and this chemical bonding reaction was kept for 72 h at 37 °C; then the covalently coated PGMA–DVB microspheres were soxhlet extracted by water to remove the residual unreacted compounds for 48 h; after that the coated
An optical contact angle measuring device (OCA20, Dataphysics, Germany) was employed to study the surface hydrophilicity of PGMA– DVB microspheres before and after coating. The contact angle of microspheres was measured by using the pressed-disk technique. The microspheres were firstly pressed into a disk by a mold, and then the water was dropped onto the disk by a needle at a speed of 0.5 μL/drop. 2.4. Determination of BSA and ovalbumin irreversible adsorption on microspheres The hydrophobic adsorption of protein solutions (5 mg/mL) of bovine serum albumin (BSA) and ovalbumin was tested on the microspheres. The PGMA–DVB microspheres coated with dextran were filled into φ4.6 × 50 mm stainless steel column by slurry packing method on a SHIMADZU LC-8A (Japan). The mobile phase was 50 mM phosphate buffer (pH 7.0 at 1 mL/min, with an injection volume of 200 μL). Repeated 5 injections of proteins were carried out on the SHIMADZU LC-8A (Japan). After each injection, the column was washed with 10 column volumes (CV) of 1.0 M NaOH for the column clean process [25,26]. All chromatography processes were performed at ambient temperature. Repeated injections of a protein should result in a constant peak area if there is no irreversible adsorption of proteins. The uncoated PGMA–DVB microsphere was also evaluated as a control batch. Super Fluor 488-labeling BSA (SF488-BSA) adsorption experiments were also conducted in a 4 °C incubator, after 24 h adsorption, the microspheres were separated from the SF488-BSA solution by centrifugation and washed with PBS 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 a TCS SP2 laser scanning confocal microscope (LCSM) (Leica, Germany) to visualize the adsorption of SF488-BSA onto microspheres. The samples were detected at 488 nm, and the fluorescent images at 520– 550 nm wavelengths were then taken.
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2.5. Measurement of microspheres pore diameter and morphology Pore diameter measurement of PGMA–DVB before and after coating was conducted by an AutoPore IV 9500 mercury porosimetry (Micromeritics, USA) to study the difference between PGMA–DVB microspheres and PGMA–DVB–dextran. Experiments were performed in accordance with the protocol given in the AutoPore IV 9500 operator's manual. The morphologies of the microsphere before and after coating were observed by scanning electron micrograph (SEM) (JSM-6400, JEOL Lim. Co., Japan). The detailed method of the sample preparations for SEM observation was described as follows. Microspheres were re-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 was coated with a thin gold film under reduced pressure below 5 Pa with a JFC-1600 fine coater (JEOL, Japan). 2.6. Mechanical stability and permeability of media The PGMA–DVB–dextran microspheres were packed into aφ4.6 × 50 mm stainless steel column as described on Chapter 2.4. The mechanical stability and permeability of the media were evaluated through measuring the backpressures of column at different flow rates, and the mobile phase was 50 mM phosphate buffer (pH 7.0).
transesterification and resulted in the direct attachment of methacryloyl groups at the dextran [23] (Scheme 1). On the basis of basicity, the order of reactivity was OH-2>OH-3> OH-4 of the glucopyranose ring [28]. However, steric hindrance at position 2 might induce substitution of OH-3. Van Dijk-Wolthuis studied the reaction of GMA with dextran in DMSO-d6 via NMR spectra and found that the substitution positions were at OH-2 and OH-3 in a 1:1 ratio [23]. Thus, the hydrophilic coating was covalently bound to the PGMA–DVB microspheres. Fig. 2 showed that the covalently bonding dextran ratio (CBDR) increased firstly then decreased at the titer range of 0.032–0.26 mol/L DMAP. DMAP, as catalyst, could accelerate the epoxy group to react with hydroxyl group of the dextran [29]. Therefore, the covalently bonding dextran ratio (CBDR) increased with increase of DMAP in the early stage. Why not increase but decrease with the rise of DMAP in a later stage? The possible cause was that DMAP was adsorbed on the PGMA–DVB microsphere surface which occupied a number of adsorption sites in the microspheres resulting in desorption of the pre-adsorbed dextran on the microspheres. Excessive DMAP exacerbated desorption of dextran from the surface of microspheres. In our previous study [13], the similar phenomenon found that the addition of excessive NaOH led to desorption of the modified agarose from the surface of PS-DVB microspheres. So, it was found that CBDR decreased with the increase of DMAP in the latter. To ensure enough dextran to be covalently bonding on the microsphere, the DMAP amounts were chosen to be 0.13 mol/L.
3. Results and discussion 3.1. Physical adsorption of dextran on macroporous PGMA–DVB microspheres The adsorption amounts of dextran with various molecule weights on PGMA–DVB microspheres were shown in Fig. 1. The adsorption quality could reach the maximum in 24 h. The adsorption quality decreased with the increase of molecule weights of the coated dextran. Dextran of larger molecule weight has bigger size than those of low molecular weights [27], so that dextran with larger molecule weight was more difficult to diffuse into the inner of PGMA–DVB microspheres than those of low molecular weights. To clothe well the hydrophobic surface, the dextran with 5 K molecular weight and 24 h-adsorption time was selected for the further coating study. 3.2. Effect of DMAP amounts on covalently bonding dextran ratio on PGMA–DVB microspheres DMAP was a basic catalyst for opening the epoxy groups to react with the adsorbed dextran. In the present of DMAP, the epoxy groups in the pore surface reacted with the hydroxyl groups of dextran via
Fig. 1. Adsorption quality of dextran with various molecular weights on PGMA–DVB microspheres. Adsorption condition: dextran/microspheres/DMSO = 1:1:20 (g/g/mL), mechanical stirring for different times at room temperature.
3.3. Effect of reaction time and temperature on covalently bonding dextran ratio on PGMA–DVB microspheres Fig. 3 showed that CBDR increased first then tended to a stable value with the reaction time. In the early stage, the amounts of epoxy groups in the PGMA–DVB were higher, which were consumed by the reaction of dextran. Therefore, the covalently coated dextran reaction rates were high and its amounts increased along with the reaction time. As the reaction was carried on, the epoxy group amounts decreased to a relatively low value, thus, little dextran could react with PGMA–DVB microspheres. Therefore, the covalently coated dextran amounts turned to be the constant value in the final reaction time. From Fig. 3, it was found that the maximum amounts of covalently coated dextran had been obtained when the reaction time reached 60 h. Fig. 4 showed the effect of temperature. CBDR showed first increase and then decrease tendency at the temperature range of 17–57 °C. Increased temperature accelerated reactive rate of dextran and microsphere [29] and CBDR increased with temperature in the early stage. However, CBDR began to decrease when temperature reached 37 °C. The possible reason was higher temperature accelerates desorption of dextran on PGMA–DVB. Generally, high temperature should not be favor increasing the adsorption quantity which resulted from higher diffusion rate caused by the elevated temperature [30–32]. In order to obtain high coating amount, the reaction temperature needed to be controlled in this reaction. From Fig. 4, the covalently coated dextran reaction rate reach maximum
Scheme 1. PGMA–DVB microspheres react with dextran at the existence of DMAP.
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Fig. 2. Effect of DMAP concentrations on the covalently bonding dextran ratio.
at 37 °C. Therefore, the reaction temperature was selected to be 37 °C.
3.4. Effect of EDGE on the hydrophilicity of covalently coated PGMA–DVB microspheres with dextran Table 1 showed effect of EDGE. The contact angle of the uncoated PGMA–DVB microsphere was 134 ° (Sample 1 #). After covalently coating, it dropped to be 78 ° (Sample 2 #). Although the hydrophilicity of the microspheres had been improved to some extent after coating dextran, this value (78°) indicated that the coating dextran could not fully shield the hydrophobic surface of the PGMA–DVB microspheres. We ever found that agarose could form a unique coating layer after crosslinkage by EDGE [13]. EDGE was also extensively applied to crosslinking many biomaterials [33,34], which had good biocompatibility. In this study, EDGE was attempted to crosslink the coating dextran for forming a uniform coat on the microsphere surface. The contact angle decreased with increase of EDGE and the lowest value dropped to 11° when the concentration of EDGE reached 0.046 mol/ L. Unfortunately, the coated microspheres were aggregated at the 0.046 mol/L EDGE. Therefore, the amounts of EDGE were controlled to be less than 0.046 mol/g dry microspheres. The coated microspheres were characterized by FTIR and shown in Fig. 5. Comparing these three microspheres, the intensity of the peak at 3457 cm −1, which was attributed to –O–H stretching vibration of hydroxyl groups, was increased as the following order: Fig. 5ab Fig. 5bb Fig. 5c (Fig. 5a: Sample 1#; Fig. 5b: Sample 2#; Fig. 5c: Sample 7#). Additionally, the intensity of the peak at 1102 cm−1, which was attributed to C–O–C stretching vibration of ester bond, was also increased according to the above order. These indicated that the dextran had been successfully covalently coated onto the PGMA–DVB microspheres.
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Fig. 4. Effect of reaction time on covalently bonding dextran ratio.
Meanwhile, it also confirmed that the coated dextran had been efficaciously crosslinked by EDGE.
3.5. Pore diameter and morphology of PGMA–DVB microspheres before and after covalently coated dextran The pore diameters of microspheres (Sample 1 # and Sample 7 #) were measured by mercury porosimetry measurements and shown in Fig. 6. The average pore diameters were slightly changed from 221 nm to 201 nm after coating. In our previous study, the modified agarose (Mw = 24,000) was used to physically coat PS-DVB microspheres and had no influence on macroporous structure of microspheres [35]. In this study, PGMA–DVB coated with dextran (Mw = 5000) could also maintain the macropores and ensure high permeability. Additionally, the morphology of PGMA-DVB microsphere before (Sample 1 #) and after covalently coated (Sample 7 #) was also observed by SEM. In Fig. 7, coating dextran was observed on the surface of coated microspheres (Fig. 7B1) compared to the smooth surface of uncoated microspheres (Fig. 7A1). Meanwhile, the macropores of microspheres after coating were still preserved and not clogged by coating dextran (Fig. 7B and B1, Sample 7 #). The macropores of PGMA–DVB microspheres were a key feature for using perfusion chromatography because the convective mass transfer was realized through macropores in the media [36]. In order to verify whether the coating layer affected the permeability of the microspheres, the backpressures were measured with different flow rates on the microspheres (Sample 1 # and Sample 7 #) and shown in Fig. 8. The backpressure of the coated PGAM–DVB with dextran (Sample 7 #) was at the range of 0.3–1.7 MPa when the flow rate changed from 1 mL/min to 8 mL/min. The back pressure of Sample 7 # was slightly increased compared to the uncoated PGMA–DVB microsphere. In the meantime, the back pressure could keep a linear
Table 1 Contact angles of different PGMA–DVB microspheres. Sample no.
Fig. 3. Effect of reaction time on covalently bonding dextran ratio.
1# 2# 3# 4# 5# 6# 7# 8#
Covalently coated dextran (mg/g dry microsphere)
0 65 65 65 65 65 65 65
EDGE
Contact angle
(mol/ L)
(°)
0 0 0.013 0.019 0.026 0.033 0.039 0.046
134 78 65 45 32 17 13 11
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Fig. 5. FTIR of PGMA–DVB microspheres, a Sample 1#; b Sample 2#; c Sample 7#.
increase as the flow rate increased in Fig. 8. This indicated that the macropore structure and mechanical strength were stable even at high flow rate [37]. From all the above tests, it can be concluded that the coated dextran could reserve the macropores and has no influence on the application of the coated microspheres in perfusion chromatography. 3.6. Evaluation of BSA and ovalbumin irreversible adsorption on microsphere The adsorption of BSA and ovalbumin on the microspheres was shown in Fig. 9. The protein mass amounts were 0.40 mg for each injection and the chromatography peak areas of the repeated 5 injections were constant in the coated PGMA–DVB microspheres (Sample 7 #). These constant peak areas confirmed almost no adsorption of proteins on Sample 7 # [38]. Also, it indicated that the coated dextran layer was stable even after repeated washing with strong basic solution (1.0 M NaOH). However, the peak areas on uncoated PGMA– DVB microspheres (Sample 1 #) were less ten times than those on Sample 7 #. Moreover, the inconstant peak areas increased with the rise of the injection numbers. These indicated that the tested proteins had a large number of irreversible adsorption onto the uncoated microsphere (Sample 1 #). It was improved that the coated dextran could clothe well the hydrophobic surface of PGMA–DVB microspheres. Additionally, the coated microspheres (Sample 7 #) were immersed into 1.0 M HCl solution for 12 h and tested its stability in acid environment. The immersed microspheres were tested using the above-mentioned BSA and ovalbumin adsorption testing and almost no adsorption of proteins on it. This indicated that the coated microspheres were stable in the pH range of 0–14. In order to observe the hydrophilicity of PGMA–DVB microsphere surfaces more straightly, the SF488-BSA adsorption experiment was carried out on Sample
Fig. 6. Pore diameter distribution of microspheres, ■Sample 7#; ●Sample 1#.
Fig. 7. SEM of PGMA–DVB microsphere, A and A1 is Sample 1#; B and B1 is Sample 7#.
1 # and Sample 7 #. As shown in Fig. 10, great fluorescent BSA was observed on Sample 1 # (Fig. 10A), while little fluorescence was detected in Sample 7 # (Fig. 10B). This comparative study provided a straight insight on increasing hydrophilicity of PGMA–DVB microsphere coated with dextran.
4. Conclusions A potential hydrophilic packing support has been developed by covalently coating macropores of PGMA–DVB microspheres with dextran. The hydrophilicity of PGMA–DVB after coated with dextran was significantly increased when the coating dextran layer was crosslinked by EDGE. The optimized EDGE concentrations were 0.039 M. After coating, the hydrophilicity and biocompatibility of the PGMA–DVB microspheres were greatly enhanced. The nonspecific adsorption of proteins was nearly reduced to none and the coating dextran layer was stable after washing with 1 M NaOH solution. The macropore structure and morphology of the microspheres were not changed after coating, and high throughput could be realized on this coated media due to its high permeability. This study provided a hydrophilic chromatographic support for different types of chromatography such as ion-exchange or affinity chromatography. After further derivation, the coated microspheres would be a prospective stationary phase in high-speed preparative protein chromatography. The further application for protein separation will be submitted to a journal in future.
Fig. 8. The backpressures at various flow rates, red cycle, Sample 7#; black square, Sample 1#.
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Fig. 9. Graph of chromatography peak area versus injection number in BSA and ovalbumin adsorption by the microspheres, ● BSA on Sample 7#; ■ Ovalbumin on Sample 7#;▲ Ovalbumin on Sample 1#;▼ BSA on Sample 1#.
[15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31]
Fig. 10. LCSM of SF488-BSA adsorption on microsphere, A, Sample 1#; B, Sample 7#.
[32] [33] [34]
Acknowledgment We would like to thank the financial support from the National Natural Science Foundation of China (No. 51103158, No. 20906094 and KSCX2-EW-G-3).
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