Please wait a minute...
Frontiers of Chemical Science and Engineering

ISSN 2095-0179

ISSN 2095-0187(Online)

CN 11-5981/TQ

Postal Subscription Code 80-969

2018 Impact Factor: 2.809

Front. Chem. Sci. Eng.    2019, Vol. 13 Issue (1) : 120-132    https://doi.org/10.1007/s11705-018-1730-y
RESEARCH ARTICLE
Fabrication of high-capacity cation-exchangers for protein adsorption: Comparison of grafting-from and grafting-to approaches
Ming Zhao1, Run Liu1, Jian Luo3, Yan Sun1,2, Qinghong Shi1,2,3()
1. Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China
2. Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, China
3. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
 Download: PDF(1590 KB)   HTML
 Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks
Abstract

In this work, we have synthesized two polymer-grafted cation exchangers: one via the grafting-from approach, in which sulfopropyl methacrylate (SPM) is grafted through atom transfer radical polymerization onto Sepharose FF (the thus resulting exchanger is referred as Sep-g-SPM), and another via the grafting-to approach, in which the polymer of SPM is directly coupled onto Sepharose FF (the thus resulting exchanger is called as Sep-pSPM). Protein adsorption on these two cation exchangers have been also investigated. At the same ligand density, Sep-g-SPM has a larger accessible pore radius and a smaller depth of polymer layer than Sep-pSPM, due to the controllable introduction of polymer chains with the regular distribution of the ligand. Therefore, high-capacity adsorption of lysozyme and γ-globulin could be achieved simultaneously in Sep-g-SPM with an ionic capacity (IC) of 308 mmol·L1. However, Sep-pSPM has an irregular chain distribution and different architecture of polymer layer, which lead to more serious repulsive interaction to proteins, and thus Sep-pSPM has a lower adsorption capacity for γ-globulin than Sep-g-SPM with the similar IC. Moreover, the results from protein uptake experiments indicate that the facilitated transport of adsorbed γ-globulin occurs only in Sep-pSPM and depends on the architecture of polymer layers. Our research provides a clear clue for the development of high-performance protein chromatography.

Keywords polymer-grafted ionic exchanger      grafting technique      protein adsorption      atom transfer radical polymerization      γ-globulin     
Corresponding Author(s): Qinghong Shi   
Just Accepted Date: 04 April 2018   Online First Date: 22 June 2018    Issue Date: 25 February 2019
 Cite this article:   
Ming Zhao,Run Liu,Jian Luo, et al. Fabrication of high-capacity cation-exchangers for protein adsorption: Comparison of grafting-from and grafting-to approaches[J]. Front. Chem. Sci. Eng., 2019, 13(1): 120-132.
 URL:  
https://academic.hep.com.cn/fcse/EN/10.1007/s11705-018-1730-y
https://academic.hep.com.cn/fcse/EN/Y2019/V13/I1/120
Fig.1  Scheme 1Preparation of “Sep-g-SPM” cation-exchangers and synthetic polymer of SPM (pSPM) via ATRP
Name Sepharose-Br /g SPM /mmol AEM /mmol Bpy /mmol CuBr2 /mmol CuBr /mmol BMPA /mmol Solvent /mL
Sep-g-SPM300 3 10.0 0.50 3.6 0.05 1.8 0.6 21
Sep-g-SPM200 3 6.5 0.33 3.6 0.05 1.8 0.6 21
Sep-g-SPM100 3 2.0 0.10 3.6 0.05 1.8 0.6 21
pSPM ? 72 3.60 3.6 0.05 1.8 1.2 24
Tab.1  Synthetic information of polymer-grafted cation exchangers and pSPM via ATRP
Name ?????????Epoxying resin /g pSPM /(g·mL?1)
Sep-pSPM100 3.0 0.7?1.0
Sep-pSPM150 3.0 1.0?2.0
Sep-pSPM200 3.0 2.0?2.5
Sep-pSPM450 3.0 3.0?4.0
Tab.2  Synthetic information of polymer-grafted cation exchangers via grafting-to approach
Name IC /(mmol·L?1) dp /µm rpb)/(g·mL?1) CNaCl = 50 mmol·L?1 CNaCl = 200 mmol·L?1
rpore /nm Layer depth/nm rpore /nm Layer depth /nm
Sepharose FF 0.0 90±2 1.013 18.7±0.6 ? 18.8±0.5 ?
SP Sepharose FFa) 180-250 90 1.028 17.1±0.7 1.6 17.3±0.6 1.5
SP Sepharose XL 288±2 108±2 1.081 8.3±0.6 10.4 8.8±0.5 10.0
Sep-g-SPM100 108±1 91±2 1.030 17.4±0.5 1.3 17.7±0.4 1.1
Sep-g-SPM200 204±3 92±2 1.066 15.7±1.1 3.0 16.5±0.9 2.3
Sep-g-SPM300 308±2 94±2 1.074 9.6±0.4 9.1 10.3±0.4 8.5
Sep-pSPM100 105±1 91±2 1.028 12.5±1.1 6.2 13.3±0.9 5.5
Sep-pSPM150 150±4 92±2 1.023 12.4±1.1 6.3 13.2±0.9 5.6
Sep-pSPM200 210±5 94±2 1.024 8.8±0.5 9.8 9.5±0.5 9.3
Sep-pSPM450 455±6 108±2 1.031 7.7±0.8 10.9 8.4±0.6 10.4
Tab.3  Physical properties of commercial and polymer-grafted cation-exchangers
Fig.2  FTIR spectra of (A) Sepharose FF, (B) Sepharose Br, (C) Sep-g-SPM300, and (D) Sep-pSPM450
Fig.3  Dextran calibration curves for Sepharose FF, SP Sepharose FF, SP Sepharose XL, Sep-g-SPM and Sep-pSPM cation exchangers. In the measurements, 50 mmol·L?1 NaCl in 20 mmol·L?1 Tris-HCl buffer (pH 8.0) was used as the liquid phase
Fig.4  Adsorption isotherms of lysozyme (a, b) and γ-globulin (c, d) on Sep-g-SPM (a, c) and Sep-pSPM (b, d) cation exchangers. Adsorption buffer is 50 mmol·L1 NaCl in 20 mmol·L1 Tris-HCl buffer (pH 8.0) for lysozyme adsorption and 50 mmol·L1 NaCl in 20 mmol·L1 acetate buffer (pH 4.5) for g-globulin adsorption. The curves in the figure are the fitted Langmuir isotherm by Eq. (1)
Name Lysozyme g-globulin
qm /(mg·mL1) Kd /(mg·mL1) De/D0 qm /(mg·mL1) Kd /(mg·mL1) De/D0
SP Sepharose FF 156.1±6.8 0.020±0.008 0.403 203.6±16.4 0.090±0.030 0.094
SP Sepharose XL 269.5±7.7 0.022±0.003 0.572 233.3±13.4 0.090±0.020 0.308
Sep-g-SPM100 86.3±2.3 0.072±0.013 0.391 111.2±3.2 0.050±0.010 0.363
Sep-g-SPM200 220.5±15.7 0.019±0.006 0.602 221.1±6.2 0.024±0.003 0.077
Sep-g-SPM300 362.9±33.9 0.023±0.007 0.130 256.9±15.8 0.090±0.030 0.033
Sep-pSPM100 56.5±2.8 0.019±0.007 0.170 66.8±1.9 0.019±0.004 0.100
Sep-pSPM150 158.8±5.1 0.013±0.002 0.181 203.3±6.0 0.027±0.003 0.033
Sep-pSPM200 324.3±18.6 0.010±0.002 0.114 177.7±28.3 0.471±0.244 0.088
Sep-pSPM450 462.4±50.3 0.012±0.004 0.051 50.7±1.6 0.233±0.041 0.099
Tab.4  Langmuir parameters and De/D0 for lysozyme and γ-globulin adsorption
Fig.5  qm for lysozyme on Sep-pSPM cation exchangers as a function of ionic capacity. The symbol solid square (n) stands for qm for lysozyme on Sep-pSPM cation exchangers, and solid circle (l) for qm for lysozyme in [6]. The straight line was obtained by fitting with all the experimental data
Name Lysozyme g-globulin
qm /(mg·mL?1) Kd /(mg·mL?1) De/D0 qm /(mg·mL?1) Kd /(mg·mL?1) De/D0
SP Sepharose FF 156.1±6.8 0.020±0.008 0.403 203.6±16.4 0.090±0.030 0.094
SP Sepharose XL 269.5±7.7 0.022±0.003 0.572 233.3±13.4 0.090±0.020 0.308
Sep-g-SPM100 86.3±2.3 0.072±0.013 0.391 111.2±3.2 0.050±0.010 0.363
Sep-g-SPM200 220.5±15.7 0.019±0.006 0.602 221.1±6.2 0.024±0.003 0.077
Sep-g-SPM300 362.9±33.9 0.023±0.007 0.130 256.9±15.8 0.090±0.030 0.033
Sep-pSPM100 56.5±2.8 0.019±0.007 0.170 66.8±1.9 0.019±0.004 0.100
Sep-pSPM150 158.8±5.1 0.013±0.002 0.181 203.3±6.0 0.027±0.003 0.033
Sep-pSPM200 324.3±18.6 0.010±0.002 0.114 177.7±28.3 0.471±0.244 0.088
Sep-pSPM450 462.4±50.3 0.012±0.004 0.051 50.7±1.6 0.233±0.041 0.099
Tab.5  Langmuir parameters and De/D0 for lysozyme and γ-globulin adsorption
Fig.6  qm for lysozyme on Sep-pSPM cation exchangers as a function of ionic capacity. The symbol solid square (n) stands for qm for lysozyme on Sep-pSPM cation exchangers, and solid circle (l) for qm for lysozyme in [6]. The straight line was obtained by fitting with all the experimental data
Fig.7  Adsorption isotherms of (a,b) lysozyme and (c,d) γ-globulin on (a,c) Sep-g-SPM300 and (b,d) Sep-pSPM450 at various salt concentrations. In the experiments, the base adsorption buffers are the same as those in Fig. 3 and salt concentrations of 0, 50, 100 and 200 mmol·L?1 were adjusted by adding NaCl
Name Proteins CNaCl /(mmol·L?1)
0 50 100 200
Sep-g-SPM300 Lysozyme qm /(mg·mL?1) 412.7±37.7 362.9±33.9 328.3±7.3 246.5±32.1
? ? Kd /(mg·mL?1) 0.009±0.002 0.023±0.007 0.033±0.003 0.645±0.203
? ? De/D0 0.090 0.130 0.301 0.542
? γ-Globulin qm /(mg·mL?1) 200.1±23.7 256.9±15.8 233.0±5.6 182.5±12.5
? ? Kd /(mg·mL?1) 1.54±0.40 0.09±0.03 0.12±0.01 3.02±0.37
? ? De/D0 0.077 0.033 0.088 1.100
Sep-pSPM450 Lysozyme qm /(mg·mL?1) 377.3±44.4 462.4±50.3 322.5±24.1 387.5±27.5
? ? Kd /(mg·mL?1) 0.008±0.002 0.012±0.004 0.008±0.003 0.626±0.103
? ? De/D0 0.059 0.051 0.108 0.481
? γ-Globulin qm /(mg·mL?1) 51.4±8.3 50.7±1.6 45.1±1.8 77.1±2.9
? ? Kd /(mg·mL?1) 2.24±0.74 0.233±0.041 0.148±0.040 0.244±0.048
? ? De/D0 ? 0.099 0.231 0.198
Tab.6  Langmuir parameters and De/D0 for lysozyme and g-globulin adsorption at various salt concentrations
Fig.8  Uptake of (a, b) lysozyme and (c, d) γ-globulin on (a, c) Sep-g-SPM300 and (b, d) Sep-pSPM450 at various salt concentrations. In the measurements, the adsorption buffers are the same as those in Fig. 3 and salt concentrations of 0, 50, 100 and 200 mmol·L?1 were adjusted by adding NaCl
Name Lysozyme γ-globulin
DBC/(mg·mL?1) DBC/qm DBC/(mg·mL?1) DBC/qm
Sep-g-SPM300 190±2 0.52 33±3 0.13
Sep-pSPM450 111±2 0.24 5±1 0.10
Tab.7  DBCs of Sep-g-SPM300 and Sep-pSPM450 cation exchangers
1 I NSavina  I Y Galaev, B Mattiasson. Ion-exchange macroporous hydrophilic gel monolith with grafted polymer brushes. Journal of Molecular Recognition, 2006, 19(4): 313–321
https://doi.org/10.1002/jmr.774
2 E XPerez-Almodovari, YWu, G Carta. Multicomponent adsorption of monoclonal antibodies on macroporous and polymer grafted cation exchangers. Journal of Chromatography. A, 2012, 1264: 48–56
https://doi.org/10.1016/j.chroma.2012.09.064
3 J EBasconi, G Carta, M RShirts. Multiscale modeling of protein adsorption and transport in macroporous and polymer-grafted ion exchangers. AIChE Journal. American Institute of Chemical Engineers, 2014, 60(11): 3888–3901
https://doi.org/10.1002/aic.14621
4 A MLenhoff. Protein adsorption and transport in polymer-functionalized ion-exchangers. Journal of Chromatography. A, 2011, 1218(49): 8748–8759
https://doi.org/10.1016/j.chroma.2011.06.061
5 M CStone, G Carta. Protein adsorption and transport in agarose and dextran-grafted agarose media for ion exchange chromatography. Journal of Chromatography. A, 2007, 1146(2): 202–215
https://doi.org/10.1016/j.chroma.2007.02.041
6 H YWang, Y Sun, S LZhang, JLuo, Q H Shi. Fabrication of high-capacity cation-exchangers for protein chromatography by atom transfer radical polymerization. Biochemical Engineering Journal, 2016, 113: 19–29
https://doi.org/10.1016/j.bej.2016.05.006
7 B DBowes, H Koku, K JCzymmek, A MLenhoff. Protein adsorption and transport in dextran-modified ion-exchange media. I: Adsorption. Journal of Chromatography. A, 2009, 1216(45): 7774–7784
https://doi.org/10.1016/j.chroma.2009.09.014
8 L LYu, S P Tao, X Y Dong, Y Sun. Protein adsorption to poly(ethylenimine)-modified Sepharose FF: I. A critical ionic capacity for drastically enhanced capacity and uptake kinetics. Journal of Chromatography A, 2013, 1305: 76–84
https://doi.org/10.1016/j.chroma.2013.07.014
9 Q HShi, G D Jia, Y Sun. Dextran-grafted cation exchanger based on superporous agarose gel: Adsorption isotherms, uptake kinetics and dynamic protein adsorption performance. Journal of Chromatography A, 2010, 1217(31): 5084–5091
https://doi.org/10.1016/j.chroma.2010.05.065
10 YTao, G Carta, GFerreira, DRobbins. Adsorption of deamidated antibody variants on macroporous and dextran-grafted cation exchangers: I. Adsorption equilibrium. Journal of Chromatography A, 2011, 1218(11): 1519–1529
https://doi.org/10.1016/j.chroma.2011.01.049
11 S LZhang, M Zhao, WYang, JLuo, Y Sun, Q HShi. A novel polymer-grafted cation exchanger for high-capacity protein chromatography: The role of polymer architecture. Biochemical Engineering Journal, 2017, 128: 218–227
https://doi.org/10.1016/j.bej.2017.10.006
12 EUnsal, B Elmas, BCaglayan, MTuncel, SPatir, ATuncel. Preparation of an ion-exchange chromatographic support by a “grafting from” strategy based on atom transfer radical polymerization. Analytical Chemistry, 2006, 78(16): 5868–5875
https://doi.org/10.1021/ac060506l
13 L LYu, Y Sun. Protein adsorption to poly(ethylenimine)-modified Sepharose FF: II. Effect of ionic strength. Journal of Chromatography A, 2013, 1305: 85–93
https://doi.org/10.1016/j.chroma.2013.07.016
14 CChang, A M Lenhoff. Comparison of protein adsorption isotherms and uptake rates in preparative cation-exchange materials. Journal of Chromatography. A, 1998, 827(2): 281–293
https://doi.org/10.1016/S0021-9673(98)00796-1
15 AStaby, I H Jensen, I Mollerup. Comparison of chromatographic ion-exchange resins I. Strong anion-exchange resins. Journal of Chromatography A, 2000, 897(1-2): 99–111
https://doi.org/10.1016/S0021-9673(00)00780-9
16 B DBowes, A M Lenhoff. Protein adsorption and transport in dextran-modified ion-exchange media. II. Intraparticle uptake and column breakthrough. Journal of Chromatography A, 2011, 1218(29): 4698–4708
https://doi.org/10.1016/j.chroma.2011.05.054
17 B DBowes, A M Lenhoff. Protein adsorption and transport in dextran-modified ion-exchange media. III. Effects of resin charge density and dextran content on adsorption and intraparticle uptake. Journal of Chromatography A, 2011, 1218(40): 7180–7188
https://doi.org/10.1016/j.chroma.2011.08.039
18 A RUbiera, G Carta. Radiotracer measurements of protein mass transfer: Kinetics in ion exchange media. Biotechnology Journal, 2006, 1(6): 665–674
https://doi.org/10.1002/biot.200600023
19 FDismer, M Petzold, JHubbuch. Effects of ionic strength and mobile phase pH on the binding orientation of lysozyme on different ion-exchange adsorbents. Journal of Chromatography. A, 2008, 1194(1): 11–21
https://doi.org/10.1016/j.chroma.2007.12.085
20 JHubbuch, T Linden, EKnieps, ALjunglof, JThommes, M RKula. Mechanism and kinetics of protein transport in chromatographic media studied by confocal laser scanning microscopy. Part I. The interplay of sorbent structure and fluid phase conditions. Journal of Chromatography A, 2003, 1021(1-2): 93–104
https://doi.org/10.1016/j.chroma.2003.08.112
21 J WChan, A Huang, K EUhrich. Self-assembled amphiphilic macromolecule coatings: Comparison of grafting-from and grafting-to approaches for bioactive delivery. Langmuir, 2016, 32(20): 5038–5047
https://doi.org/10.1021/acs.langmuir.6b00524
22 CReznik, C F Landes. Transport in supported polyelectrolyte brushes. Accounts of Chemical Research, 2012, 45(11): 1927–1935
https://doi.org/10.1021/ar3001537
23 SHansson, V Trouillet, TTischer, A SGoldmann, ACarlmark, CBarner-Kowollik, EMalmstrom. Grafting efficiency of synthetic polymers onto biomaterials: A comparative study of grafting-from versus grafting-to. Biomacromolecules, 2013, 14(1): 64–74
https://doi.org/10.1021/bm3013132
24 SMinko. Grafting on solid surfaces: “Grafting to” and “grafting from” methods. In: Stamm M, ed. Polymer Surfaces and Interfaces: Characterization, Modification and Applications. Berlin: Springer Berlin Heidelberg, 2008, 215–234
25 .          Z GWang, L SWan, Z KXu. Surface engineerings of polyacrylonitrile-based asymmetric membranes towards biomedical applications: An overview. Journal of Membrane Science, 2007, 304(1-2): 8–23
https://doi.org/10.1016/j.memsci.2007.05.012
26 L LYu, Q H Shi, Y Sun. Effect of dextran layer on protein uptake to dextran-grafted adsorbents for ion-exchange and mixed-mode chromatography. Journal of Separation Science, 2011, 34(21): 2950–2959
https://doi.org/10.1002/jssc.201100394
27 Q HShi, X Zhou, YSun. A novel superporous agarose medium for high-speed protein chromatography. Biotechnology and Bioengineering, 2005, 92(5): 643–651
https://doi.org/10.1002/bit.20652
28 L EWeaver, G Carta. Protein adsorption on cation exchangers: comparison of macroporous and gel-composite media. Biotechnology Progress, 1996, 12(3): 342–355
https://doi.org/10.1021/bp960021q
29 PDephillips, A M Lenhoff. Pore size distributions of cation-exchange adsorbents determined by inverse size-exclusion chromatography. Journal of Chromatography A, 2000, 883(1-2): 39–54
https://doi.org/10.1016/S0021-9673(00)00420-9
30 QLi, J Imbrogno, GBelfort, X LWang. Making polymeric membranes antifouling via “grafting from” polymerization of zwitterions. Journal of Applied Polymer Science, 2015, 132(21): n/a
https://doi.org/10.1002/app.41781
31 FDismer, J Hubbuch. A novel approach to characterize the binding orientation of lysozyme on ion-exchange resins. Journal of Chromatography. A, 2007, 1149(2): 312–320
https://doi.org/10.1016/j.chroma.2007.03.074
32 S H SKoshari, N JWagner, A MLenhoff. Effects of resin architecture and protein size on nanoscale protein distribution in ion-exchange media. Langmuir, 2018, 34(2): 673–684
https://doi.org/10.1021/acs.langmuir.7b03289
33 HYang, P V Gurgel, R G Carbonell. Purification of human immunoglobulin G via Fc-specific small peptide ligand affinity chromatography. Journal of Chromatography A, 2009, 1216(6): 910–918
https://doi.org/10.1016/j.chroma.2008.12.004
34 B DFair, A M Jamieson. Studies of protein adsorption on polystyrene latex surfaces. Journal of Colloid and Interface Science, 1980, 77(2): 525–534
https://doi.org/10.1016/0021-9797(80)90325-2
35 JLuo, Y Wan. Effect of highly concentrated salt on retention of organic solutes by nanofiltration polymeric membranes. Journal of Membrane Science, 2011, 372(1-2): 145–153
https://doi.org/10.1016/j.memsci.2011.01.066
36 C  A Yoshikawa, YGoto, TTsujii, TFukuda, KKimura, AYamamoto, Kishida. Protein repellency of well-defined, concentrated poly(2-hydroxyethyl methacrylate) brushes by the size-exclusion effect. Macromolecules, 2006, 39(6): 2284–2290
https://doi.org/10.1021/ma0520242
[1] Shenggang Chen, Tao Liu, Ruiqi Yang, Dongqiang Lin, Shanjing Yao. Preparation of copolymer-grafted mixed-mode resins for immunoglobulin G adsorption[J]. Front. Chem. Sci. Eng., 2019, 13(1): 70-79.
[2] LI Aixiang, LU Zaijun, LÜ Zijian. Synthesis and characterization of well-defined comb-like branched polymers [J]. Front. Chem. Sci. Eng., 2008, 2(4): 407-411.
[3] ZHOU Xiaopeng, SU Xueli, SUN Yan. Analysis of statistical thermodynamic model for binary protein adsorption equilibria on cation exchange adsorbent[J]. Front. Chem. Sci. Eng., 2007, 1(2): 103-112.
[4] YU Tao, WANG Yun, LU Dairen, BAI Ruke, LU Weiqi. Synthesis of mid-dicarboxy polystyrene by ATRP and formation of ionic-bonded supramolecules[J]. Front. Chem. Sci. Eng., 2007, 1(2): 140-145.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed