Purification of Antibodies by Affinity Chromatography

Year 2011, Volume: 39 Issue: 1, 1 - 18, 01.01.2011

Adil Denizli

Abstract

Antibodies are becoming a dominant protein class for various human therapeutical applications and they represent the largest number of molecules in clinical trials today. Antibodies are purified using a combination of various purification techniques. The most popular technique is affinity chromatography based on specific interaction of immunoglobulin G IgG with affinity ligand. Affinity chromatography has several advantages since it is an easy, fast and selective procedure for capturing the target antibody. This review aims at describing different affinity techniques for purification of antibodies and reports the contributions of scientists working in this field and their important results. The main issues discussed in this review are: protein A affinity chromatography, histidine-ligand affinity chromatography, immobilized metal-chelate affinity chromatography, dye-ligand affinity chromatography, biomimetic affinity chromatography, negative chromatography, mixed-mode affinity chromatography, thiophilic chromatography, magnetically stabilized fluidized beds and aqueous two phase systems.

Keywords

Affinity chromatography, immunoglobulins, antibodies, antibody purification

Afinite Kromatografisi ile Antibadi Saflaştırılması

Abstract

Antibadiler tedavi amaçlı uygulamalarda yoğun olarak kullanılan bir protein türüdür. Antibadiler değişik saflaştırma tekniklerinin bir arada kullanımı ile saflaştırılırlar. En yaygın kullanılan saflaştırma tekniği immunoglobulin G’nin IgG afinite ligand ile seçici etkileşimine dayanan afinite kromatografi tekniğidir. Afinite kromatografisinin kolay, hızlı ve hedef molekülü seçici olarak yakalama gibi birçok avantajları söz konusudur. Bu derleme makalede son yıllarda antibadi saflaştırma amacıyla kullanılan farklı afinite teknikleri ve elde edilen önemli sonuçları verilmiştir. Bu kapsamda temel ayırma teknikleri olarak; protein afinite kromatografisi, boyaligand afinite kromatografisi, immobilize metal-şelat afinite kromatografisi, karışık-mod afinite kromatografisi, tiyofilik kromatografi, manyetik olarak kararlaştırılmış akışkan yataklar ve sulu iki fazlı sistemler ve önemli sonuçları tartışılmıştır

Keywords

Afinite kromatografisi, immunoglobulinler, antibadiler, antibadi saflaştırılması

References

• [1] M. Labib, M. Hedström, M. Amin, B. Mattiasson, A multipurpose capacitive biosensor for assay and quality control of human IgG, Biotechnol. Bioeng., 104 (2009) 312.
• [2] J.M. Reichert, C.J. Rosesweig, L.B. Faden, M.C. Dewitz, Monoclonal antibody successes in the clinic, Nature Biotechnol., 23 (2005) 1073.
• [3] L. Uzun, R. Say, S. Ünal, A. Denizli, Production of surface plasmon resonance based assay-kit for hepatitis diagnosis, Biosens. Bioelectron., 24 (2009) 2878.
• [4] P.A.J. Rosa, I.F. Ferreira, A.M. Azevedo, M.R. AiresBarros, Aqueous two-phase systems: a viableplatform in the manufacturing of biopharmaceuticals, J. Chromatogr. A, 1217 (2010) 2296.
• [5] P.J. Carter, Potent antibody therapeutics by design, Nature Rev. Immunol., 6 (2006) 343.
• [6] A.A. Shukla, J. Thömmes, Recent advances in largescale production of monoclonal antibodies and related proteins, Trends in Biotechnol., 28 (2010) 253.
• [7] D. Low, R. O’Leary, N.S. Pujar, Future of antibody purification, J. Chromatogr. B., 848 (2007) 48.
• [8] L. Wang, K.Z. Mah, R. Ghosh, Purification of human IgG using membrane based hybrid bioseparation technique and its variants: a comparative study, Sep. Purif. Technol., 66 (2009) 242.
• [9] M. Wilchek, My life with affinity, Protein Science, 13 (2004) 3066.
• [10] P. Cuatrecasas, M. Wilchek, C.B. Anfinsen, Selective enzyme purification by affinity chromatography, Proc. Natl. Acad. Sci. USA, 61 (1968) 636.
• [11] J. Turkova, Bioaffinity Chromatography, Elsevier, Amsterdam, 1993.
• [12] P. Matejtschuk, (Ed), Affinity Separations, A Practical Approach, IRL Press, Oxford, 1997.
• [13] M.P. Deutscher, (Ed) Guide to Protein Purification, Methods in Enzymology, Volume 182, Academic Press, San Diego, 1990.
• [14] A. Denizli, E. Pişkin, E., DNA immobilized polyhydroxyethylmethacrylate microbeads for affinity sorption of human IgG and anti-DNA antibodies, J. Chromatogr. B, 666 (1995) 215.
• [15] Y.B. Yang, K. Harrison, Influence of column type and chromatographic conditions on the ionexchange chromatography of immunoglobulins, J. Chromatogr. A., 743 (1996) 171.
• [16] G.A. Perry, J.D. Jackson, T.L. McDonald, D.A. Crouse and J.G. Sharp, Purification of monoclonal antibodies using HPLC, Prep. Biochem., 14 (1984) 431.
• [17] B. Pavlu, U. Johansson, C. Nyhlen, A. Wichman, Rapid purification of monoclonal antibodies by HPLC, J. Chromatogr., 359 (1986) 449.
• [18] S.W. Burchiel, J.R. Billman, T.R. Alber, Rapid and efficient purification of mouse monoclonal antibodies from ascites fluid using HPLC, J. Immunol. Methods, 69 (1984) 33.
• [19] A.C.A. Roque, C.S.O. Silva, M.A. Taipa, Affinity-based methodologies and ligands for antibody purification: advances and perspectives, J. Chromatogr. A., 1160 (2007) 44.
• [20] M.G. Gore, W.F. Ferris, A.G. Popplewell, M. Scaven, T. Atkinson, Protein Eng., 5 (1992) 577.
• [21] A. Denizli, Y. Arıca, Protein A immobilized microporous PHEMA affinity membranes for selective sorption of IgG from human plasma, J. Biomater. Sci. Polym. Ed., 11 (2000) 367.
• [22] J.J. Langone, Applications of immobilized protein A in immunochemical techniques, J. Immunol. Methods, 55 (1982) 277.
• [23] P. Füglistaller, Comparison of immunoglobulin binding capacities and ligand leakage using eight different protein A affinity matrices, J. Immunol. Methods, 124 (1989) 171.
• [24] P.R. Hari, W. Paul, C.P. Sharma, Adsorption of human IgG on Cu(II)-immobilized cellulose affinity membrane: preliminary study, J. Biomed. Mater. Res., 50 (2000) 110.
• [25] M. Linhult, S. Gulich, T. Graslund, A. Simon, M. Karlsson, A. Sjoberg, K. Nord, S. Hober, Proteins, 55 (2004) 407.
• [26] BE Helatcare, Downstream, vol. 39, 2005.
• [27] C. Boi, S. Dimartino, G.C. Sarti, Performance of new protein A affinity membranee for the primary recovery of antibodies, Biotechnol. Prog., 24 (2008) 640.
• [28] Z. Ma, S. Ramakrishna, Electrospun regenerated cellulose nanofiber affinity membrane functionalized with protein A/G for IgG purification, J. Membr, Sci., 319 (2008) 23.
• [29] A. Denizli, E. Pişkin, J. Chromatogr. B., Protein A immobilized PHEMA beads for affinity sorption of human IgG, 668 (1995) 13.
• [30] E. Klein, D. Yeager, R. Seshadri, U. Baurmeister, Affinity adsorption devices prepared from microporous poly(amide) hollow fibers and sheet membranes, J. Membr. Sci., 129 (1997) 31.
• [31] E. Klein, E. Eichholz, D.H. Yeager, Affinity membrane prepared from hydrophilic coatings on microporous polysulfone hollow fibers, J. Membr. Sci., 90 (1994) 69.
• [32] P. Langlotz, K.H. Kroner, Surface modified membranes as a matrix for protein purification, J. Chromatogr. A, 591 (1992) 107.
• [33] O.P. Dancette, K.L. Taboureau, E. Tournier, C. Charcosset, P. Blond, Purification of IgG by protein A/G affinity membrane chromatography, J. Chromatogr. B., 723 (1999) 61.
• [34] L.R. Castilho, W.D. Deckwer, F.B. Anspach, Influence of matrix activation and polymer coating on the purification of human IgG with protein A affinity membranes, J. Membr. Sci., 172 (2000) 269.
• [35] H. Alkan, N. Bereli, Z. Baysal, A. Denizli, Antibody purification with protein A attached supermacroporous PHEMA cryogel, Biochem. Eng. J., 45 (2009) 201.
• [36] E. Klein, E. Eichholz, F. Theimer, D. Yeager, Chitosan modified sulfonated poly(ethersulfone) as a support for affinity separations, J. Membr. Sci., 95 (1994) 199.
• [37] K.C. Hou, R. Zaniewski, S. Roy, Protein A immobilized affinity cartridge for IgG purification, Biotechnol. Appl. Biochem., 13 (1991) 257.
• [38] L. Uzun, D. Türkmen, V. Karakoç, H. Yavuz, A. Denizli, Performance of protein A based affinity membranes for antibody purification, J. Biomater. Sci., 2010, in press.
• [39] Q.Luo, H. Zou, Q. Zhang, X. Xiao, J. Ni, High performance affinity chromatography with immobilization of protein A and L-histidine on molded monolith, Biotechnol. Bioeng., 80 (2002) 481.
• [40] L. Jia, L. Yang, H. Zou, Y. Zhang, J. Zhao, C. Fan, L. Sha, Protein A tangential flow affinity membrane cartridge for extracorpore-al immunoadsorption therapy, Biomed. Chromatogr., 13 (1999) 472.
• [41] S. Ghose, D. Nagrath, B. Hubbard, C. Brooks, S.M. Cramer, Use and optimization of a dual-flow rate loading strategy to maximize throughput in protein A affinity chromatography, Biotechnol. Prog., 20 (2004) 830.
• [42] X. Liu, Y. Guan, Y. Yang, Z. Ma, X. Wu, H. Liu, Preparation of superparamagnetic immuno microspheres and application for antibody purification, J. Appl. Polym. Sci., 94 (2004) 2205.
• [43] L. Yang, P. Chen, Chitosan/coarse filter paper composite membrane for fast purification of IgG from human serum, J. Membr. Sci., 205 (2002) 141.
• [44] C.H. Bamford. K.G. Al-Lamee, M.D. Purbrick, T.J. Wear, Studies of a novel membrane for affinity separations I: functionalisation and protein coupling, J. Chromatogr., 606 (1992) 19.
• [45] Z. Ma, Z. Lan, T. Matsuura, S. Ramakrishna, Electrospun polyethersulfone affinity membrane: preparation and performance evaluation, J. Chromatogr. B., 877 (2009) 3686.
• [46] H. Zou, Q. Luo, D. Zhou, Affinity membrane chromatography for the analysis and purification of proteins, J. Biochem. Biophys. Methods, 49 (2001) 199.
• [47] E.B. Altıntaş, A. Denizli, Monosize magnetic hydrophobic beads for lysozyme purification under magnetic field, Mater. Sci. Eng. C, 29 (2009) 1627.
• [48] D.A. Uygun, M.E. Çorman, N. Öztürk, S. Akgöl, A. Denizli, Poly(hydroxyethyl methacrylate-methacryloyl amidotryptophane) nanospheres and their utilization as affinity adsorbents for lipase adsorption, Mater. Sci. Eng. C, 30 (2010) 1285.
• [49] M.A. Vijayalakshmi, Pseudobiospecific ligand affinity chromatography, Trends Biotechnol., 7 (1989) 71.
• [50] M.A. Vijayalakshmi, Histidine ligand affinity chromatography, Mol. Biotechnol., 6 (1996) 347.
• [51] R. Bhattacharyya, R.P. Saha, U. Samana, P. Chakrabarti, J. Proteome Res., 2 (2003) 255.
• [52] A. Elkak, S. Ismail, L. Uzun, A. Denizli, Adsorption study of IgG subclasses from different species by pseudobioaffinity separation on histidyl-bisoxiraneSepharose, Chromatographia, 69 (2009) 1161.
• [53] Y. Çanak, S. Özkara, S. Akgöl, A. Denizli, Pseudospecific bioaffinity chromatography of IgG, React. Functl. Polym., 61 (2004) 369.
• [54] S. Akgöl, S. Özkara, L. Uzun, F. Yılmaz, A. Denizli, Pseudospecific magnetic affinity beads for IgG depletion from human serum, J. Appl. Polym. Sci., 106 (2007) 2405.
• [55] S. Özkara, B. Garipcan, E.Pişkin, A, Denizli, MAH carrying pseudospecifc affinity sorbent for IgG isolation from human plasma in column system, J. Biomater. Sci. Polym. Ed., 14 (2003) 761.
• [56] B. Garipcan, A. Denizli, A novel chromatographic affinity support material for separation of IgG from human plasma, Macromol. Biosci., 2 (2002) 135.
• [57] L. Uzun, R. Say, A. Denizli, Porous PHEMA based monolith as a new adsorbent for affinity chromatography, React. Functl. Polym., 64 (2005) 93.
• [58] R.D. Arrua, M.C. Strumia, C.I.A. Igarzabal, Macroporous monolithic polymers: preparation and applications, Materials, 2 (2009) 2429.
• [59] H. Sun, L. Zhang, H. Chai, J. Yu, H. Qian, H. Chen, A study of human γ-globulin adsorption capacity of PVDF hollow fiber affinity membranes containing different amino acid ligands, Sep. Purif. Technol., 48 (2006) 281.
• [60] S. Özkara. H. Yavuz, S. Patır, M.Y. Arıca, A. Denizli, Separation of HIgG with L-histidine immobilised pseudo-specific bioaffinity adsorbents, Sep. Sci. Technol., 37 (2002) 717.
• [61] N. Öztürk, A. Tabak, S. Akgöl, A. Denizli, Newly synthesized bentonite-histidine (bent-his) microcomposite affinity sorbents for IgG adsorption, Colloids Surf. A., 301 (2007) 490.
• [62] N. Öztürk, M.E. Günay, S. Akgöl, A. Denizli, Silanemodified magnetic beads: application to IgG separation, Biotechnol. Prog., 23 (2007) 1149.
• [63] N. Öztürk, N. Bereli, S. Akgöl, A. Denizli, High capacity binding of antibodies by PHEMA nanoparticles, Colloids Surf. B., 67 (2008) 14.
• [64] D. Türkmen, N. Öztürk, A. Elkak, S. Akgöl, A. Denizli, Phenylalanine containing hydrophobic nanospheres for antibody purification, Biotechnol. Prog., 24 (2008) 1297.
• [65] S. Özkara, S. Akgöl, Y. Çanak, A. Denizli, A novel magnetic adsorbent for IgG purification in MSFB, Biotechnol. Prog., 20 (2004) 1169.
• [66] K. Haupt, S.M.A. Bueno, M.A. Vijayalakshmi, Interaction of IgG with L-histidine immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes, J. Chromatogr. B., 674 (1995) 13.
• [67] S.M.A. Bueno, K. Haupt, M.A. Vijayalakshmi, Separation of IgG from human serum by pseudobioaffinity chromatography using immobilized L-histidine in hollow fibre membranes, J. Chromatogr. B., 667 (1995) 57.
• [68] T.S. Hwang, J.W. Park, Preparation of modified hollow propylene membrane and their adsorption properties of g-globulins, Macromol. Res., 11 (2003) 347.
• [69] M. Kim, K. Saito, S. Furusaki, T. Sugo, I. Ishigaki, Protein adsorption capacity of a porous phenylalanine containing membrane based on a polyethylene matrix, J. Chromatogr., 586 (1991) 27.
• [70] C.I. Alvarez, M.C. Trumia, H.E. Bertorello, Preparation of adsorbents applicable to pseudobiospecific ligand affinity chromatography using different spacers and ligands, React. Functl. Polym., 34 (1997) 103.
• [71] A. El-Kak, S. Manjini, V. Vijayalakshmi, Interaction of IgG with immobilized histidine: mechanistic and kinetic aspects, J. Chromatogr. 604 (1992) 29.
• [72] K. Mondal, M.N. Gupta, The affinity concept in bioseparation: evolving paradgms and expanding range of applications, Biomolular Eng., 23 (2006) 59.
• [73] R. Gutierrez, E.M. Martin del Valle, M.A. Galan, Immobilized metal-ion affinity charomatography: status and trends, Sep. Purif. Rev., 36 (2007) 71.
• [74] A. Denizli, M. Alkan, B. Garipcan, S. Özkara, E. Pişkin, Novel metal-chelate affinity adsorbent for purification of IgG from human plasma, J. Chromatogr. B, 795 (2003) 93.
• [75] R.R. Prasanna, M.A. Vijayalakshmi, Characterization of metal chelate methacrylate monolithic disk for purification of polyclonal and monoclonal IgG, J. Chromatogr. B., 1217 (2010) 3660.
• [76] S. Ozkara, H. Yavuz, A. Denizli, Purification of IgG from human plasma by metal-chelate affinity chromatography, J. Appl. Polym. Sci., 89 (2003) 1576.
• [77] N. Bereli, L. Uzun, H. Yavuz, A. Elkak, A. Denizli, Antibody purification using porous metal chelated monolithic columns, J. Appl. Polym. Sci., 101 (2006) 395.
• [78] E.B. Altıntaş, N. Tüzmen, L. Uzun, A. Denizli, Immobilized metal affinity adsorption for antibody depletion from human serum with monosize beads, Ind. Eng. Chem. Res., 46 (2007) 7802.
• [79] K. Kracalikova, M. Bleha, Chelating polymer-based membranes, preparation and use for metal ion scavenging and sorption of murine IgG by immbilized Ni(II) ions, Polym. Bull., 61 (2008) 143.
• [80] G. Tishchenko, J. Dybal, K. Meszarosova, Z. Sedlakova, M. Bleha, Purification of the specific IgG1 by IMAC using nickel complexes of chelating porous and nonporous polymeric sorbents based on poly(methacrylic esters), J. Chromatogr. A., 954 (2002) 115.
• [81] P. Pikryl, D. Horak, M. Ticha, Z. Kucerova, polymer microspheres for IMAC protein separation, J. Sep. Sci., 29 (2006) 2541.
• [82] M.B. Ribeiro, M. Vijayalakshmi, D. Todorova-Balvay, S.M.A. Bueno, Effect of IDA and TREN chelating agents and buffer systems on the purification of human IgG with immobilized nickel affinity membranes, J. Chromatogr. B., 861 (2008) 64.
• [83] M. Karataş, S. Akgöl, H. Yavuz, R. Say, A. Denizli, IgG depletion from human serum with metal chelated beads under magnetic field, Int. J. Biol. Macromol., 40 (2007) 254.
• [84] K. Kracalikova, G. Tishchenko, M. Bleha, Effect of the matrix structure and concentration of polymerimmobilized Ni2+-iminodiacetic acid complexes on retention of IgG1, J. Biochem. Biophys. Methods, 67 (2006) 7.
• [85] V. Boden, J.J. Winzerling, M. Vijayalakshmi, J. Porath, Rapid one-step purification of goat IgGs by IMAC, J. Immunol. Methods, 181 (1995) 225.
• [86] S. Jain, M.N. Gupta, Purification of goat IgG by immobilized metal-ion affinity using cross-linked alginate beads, Biotechnol. Appl. Biochem., 39 (2004) 319.
• [87] N. Bereli, G. Şener, E.B. Altıntaş, H. Yavuz, A. Denizli, Poly(glycidyl methacrylate) beads embedded cryogels for pseudo-specific affinity depletion of albumin and immunoglobulin G, Mater. Sci. Eng., 30 (2010) 323.
• [88] N. Demiryas, N. Tuzmen, I.Y. Galaev, E. Piskin, A. Denizli, Poly(acrylamide-allyl glycidyl ether) cryogel as a stationary phase in dye affinity chromatography, J. Appl. Polym. Sci., 105 (2007) 1808.
• [89] C. Babaç, H. Yavuz, I.Y. Galaev, E. Pişkin, A. Denizli, Binding of antibodies to concanavalin A-modified monolithic cryogel, React. Functl. Polym., 66 (2006) 1263.
• [90] M. Andac, F.M. Plieva, A. Denizli, I.Y. Galaev, B. Mattiasson, Poly(hydroxyethyl methacrylate)-based macroporous hydrogels with disulfide cross-linker, Macromol. Chem. Physics, 209 (2008) 577.
• [91] N. Bereli, M. Andac, G. Baydemir, R. Say, I.Y. Galaev, A. Denizli, Protein recognition via ion-coordinated molecularly imprinted supermacroporous cryogels, J. Chromatogr. A, 1190 (2008) 18.
• [92] K. Yao, S. Shen, J. Yun, L. Wang, F. Chen, X. Yu, Protein adsorption in supermacroporous cryogels with embedded nanoparticles, Biochem. Eng. J., 36 (2007) 139.
• [93] K. Yao, J. Yun, S. Shen, L. Wang, X. He, X. Yu, Characterization of a novel continuous supermacroporous monolithic cryogel embedded with nanoparticles for protein chromatography, J. Chromatogr. A., 1109 (2006) 103.
• [94] M. Le Noir, F. Plieva, T. Hey, B. Guieyse, B. Mattiasson, Macroporous molecularly imprinted polymer/ cryogel composite systems for the removal of endocrine disrupting trace contaminants, J. Chromatogr. A., 1154 (2007) 158.
• [95] A. Denizli, E. Pişkin, Dye-ligand affinity systems, J. Biochem. Biophys. Methods, 49 (2001) 391.
• [96] P.G. Byfield, S. Copping, W.A. Barlett, Fractionation of IgG by chromatography on Remazol yellow GGLsepharose, Biochem. Soc Trans., 10 (1982) 104.
• [97] R. Wongchuphan, B.T. Tey, W.S. Tan, F.S. Taip, S.M.M. Kamal, T.C. Ling, Application of dye-ligands affinity adsorbent in capturing of rabbit IgG, Biochem. Eng. J., 45 (2009) 232.
• [98] I. Roith, Essential Immunology, Ninth edition, Blackwell Science, Oxford, UK, 1997.
• [99] M. Hasnaoui, J.P. Debbia, P. Lambin, O. Bertrand, Screening of a large number of dyes for the separation of HIgG2 from the other IgG subclasses: IgG2 enrichment on immobilized procion yellow HE4R, J. Chromatogr. B., 766 (1997) 49.
• [100] H. Yavuz, S. Akgöl, R. Say, A. Denizli, Affinity separation of IgG subclasses on dye attached PHPMA beads, Int. J. Biol. Macromol., 39 (2006) 303.
• [101] J. Curling, Plasma Product Biotechnology Meeting, Curaçao, Netherlands, Antilles, 2003.
• [102] G. Fassina, A. Verdoliva, G. Polimbo, M. Ruvo, O. Cassani, Immunoglobulin specificity of TG19318: a novel synthetic ligand for antibody purification, J. Mol. Recog., 11 (1998) 128.
• [103] S.F. Teng, K. Sproule, A. Husain, C.R. Lowe, Affinity chromatography on immobilized biomimetic ligands synthesis, immobilization and chromatographic assessment of an IgG-binding ligand, J. Chromatogr. B., 740 (2000) 1.
• [104] S. Chhatre, R. Francis, N.J. Titchener-Hooker, A.R. Newcombe, E. Keshawarz-Moore, Evaluation of a novel agarose-based synthetic ligand adsorbent for therecovery of antibodies from ovine serum, J. Chromatogr. B., 860 (2007) 209.
• [105] S.M.A. Bueno, M.B. Ribeiro, J.R. Caro, F.P. dos Santos, M.P. de Castro, S.M.A. Bueno, Adsorption of human serum proteins onto TRENagarose: purification of human IgG by negative chromatography, J. Chromatogr. B., 877 (2009).
• [106] I.T.L. Bresolin, M.C.M. de Souza, S.M.A. Bueno, A new process of IgG purification by negative chromatography: Adsorption aspects of human serum proteins onto w-aminodecyl-agarose, J. Chromatogr. B., 878 (2010) 2087.
• [107] M.C.M. de Souza, I.T.L. Bresolin, S.M.A. Bueno, Purification of human IgG by negative chromatography on w-aminohexyl-agarose, J. Chromatogr. B., 878 (2010) 557.
• [108] I. Safarik, M. Safarikova, Magnetic techniques for the isolation and purification of proteins and peptides, BioMag. Res. Technol., 2 (2004) 1.
• [109] I. Safarik, M. Safarikova, Use of magnetic techniques for the isolation of cells, J. Chromatogr. B., 722 (1999) 33.
• [110] D. Horak, M. Babic, H. Mackova, M.J. Benes, Preparation and properties of magnetic nanoand microsized particles for biological and environmental separations, J. Sep. Sci., 30 (2007) 1751.
• [111] J. Hristov, L. Fachikov, An overview of separation by magnetically stabilized beds: state of the art and potential applications, China Particuol., 5 (2007) 11.
• [112] J. Hristov, Magnetic field assisted fluidization-a unified approach. part 8. mass transfer: magnetically assisted bioprocesses, Rev. Chem. Eng., 26 (2010) 55.
• [113] A. Jungbauer, Chromatographic media for bioseparation, J. Chromatogr. A., 1165 (2005) 3.
• [114] A.C.A. Roque, C.S.O. Silva, M.A. Taipa, Affinity-based methodologies and ligands for antibody purification: advances and perspectives, J. Chromatogr. A., 1160 (2007) 44.
• [115] M.J. Benes, D. Horak, F. Svec, Methacrylate based chromatographic media, J. Sep. Sci., 28 (2005) 1855.
• [116] S. Özkara, S. Akgöl, Y. Çanak, A. Denizli, A novel magnetic adsorbent for IgG purification in MSFB, Biotechnol. Prog., 20 (2004) 1169.
• [117] N.M. Hooper, Membrane biology: Do glycolipid microdomain really exist ? Curr. Biol., 8 (1998) 114.
• [118] S.V. Evans, C.R. MacKenzier, Characterization of protein-glycolipid recognition at the membrane bilayer, J. Mol. Recognit., 12 (1999) 155.
• [119] J.H. Im, T. Nakane, H. Yanagishita, T. Ikegami,. D. Kitamoto, Mannosylerythritol lipid, a yeast extracellular glycolipid, shows high binding affinity towards IgG, BMC Biotechnol., 1 (2001) 5.
• [120] J. Porath, F. Maisano, M. Belew, FEBS Lett., Thiophilic adsorption a new method for protein fractionation, 185 (1985) 306.
• [121] T.W. Hutchens, J. Porath, Thiophilic adsorption of immunoglobulins-Analysis of conditions optimal for selective immobilization and purification, Anal. Biochem., 159 (1986) 217.
• [122] H. Qian, C. Li, Z.Y. Lin, Y.X. Zhang, Using thiophilic magnetic beads in purification of antibodies from human serum, Colloids Surf. B., 75 (2010) 342.
• [123] H. Qian, C. li, Y.X. Zhang, Z.Y. Lin, Efficient isolation of IgG by paramagnetic polymer beads modified with 2-mercapto-4-mythyl-pyrimidine, J. Immunol. Methods, 343 (2010) 119.
• [124] H. Qian, Z.Y. Lin, H.M. Xu, M. Chen, The efficient and specific isolation of the antibodies from human serum by thiophilic paramagnetic polymer nanospheres, Biotechnol. Prog., 25 (2009) 376.
• [125] E. Boschetti, The use of thiophilic chromatography for antibody purification: a review, J. Biochem. Biophys. Methods, 49 (2001) 361.
• [126] R.J. Noel, W.T. O’Hare, G. Street, Thiophilic nature of divinylsulphone cross-linked agarose, J. Chromatogr. A., 734 (1996) 241.
• [R2] Q.H. Shi, Z. Cheng, Y. Sun, 4-(1H-imidazol-1-yl) aniline: a new ligand of mixed-mode chromatography for antibody purification, J. Chromatogr. A., 1216 (2009) 6081.
• [127] P. Girot, E. Averty, I. Flayeux, E. Boschetti, 2-Mercapto-5-benzimidazolesulfonic acid: an effective multimodal ligand for the separation of antibodies, J. Chromatogr. B., 808 (2004) 25.
• [128] Y. Coffinier, M.A. Vijayalakshmi, Mercaptoheterocyclic ligads grafted on a poly(ethylene vinyl alcohol) membrane for the purification of immunoglobulin G in a salt independent thiophilic chromatography, J. Chromatogr. B., 808 (2004) 51.
• [129] Y. Coffinier, C. Legallais, M.A. Vijayalakshmi, Separation of IgG from human plasma using thiophilic hollow fiber membranes, J. Membr. Sci., 208 (2002) 13.
• [130] O. Pitiot, M.A. Vijayalakshmi, in: M.A. Vijayalakshmi (ed), Biocharomatography-Theory and Practice, Taylor and Francis, London, 2002.
• [131] J. Hardouin, M. Duchateau, L. Canelle, C. Vlieghe, R.J. Caron, M. Caron, Thiophilic adsorption revisited, J. Chromatogr. B., 845 (2007) 226.
• [132] H. Qian, Z.Y. Lin, H. Ming, M.Q. Chen, The efficient and specific isolation of the antibodies from human serum by thiophilic paramagnetic polymer nanospheres, Biotechnol. Prog., 25 (2009) 376.
• [133] B. Nopper, F. Kohen, M. Wilchek, A thiophilic adsorbent for the one step high performance liquid chromatography purification of monoclonal antibodies, Anal. Biochem., 180 (1989) 66.
• [134] A. Lihme, P.M.H. Heegaard, Thiophilic adsorption chromatography: the separation of serum proteins, Anal. Biochem., 192 (1991) 64.
• [135] K.L. Knudsen, M.B. Hansen, L./R. Henriksen, B.K. Andersen, A. Lihme, Sulfone-aromatic ligands for thiophilic adsorption chromatography: purification of human and mouse IgG, Anal. Biochem., 201 (1992) 170.
• [136] A. Schwarz, F. Kohen, M. Wilchek, Novel heterocyclic ligands for the thiophilic purification of antibodies, J. Chromatogr. B., 664 (1995) 83.
• [137] S.J. Mountford, E.M. Campi, A.J. Robinson, M.T.W. Hearn, Synthesis of N-heterocyclic ligands for use in affinity and mixed mode chromatography, Tetrahedron, 67 (2011) 471.
• [138] A. Avezedo, Chromatography-free recovery of biopharmaceuticals through aqueous two-phase processing, Trends Biotechnol., 27 (2009) 240.
• [139] P.A. Albertsson, Parttion of cell particles and macromolecules, 3rd ed., Wiley New York, 1986.
• [140] A.M. Azevedo, A.G. Gomes, P.A.J. Rosa, I.F. Ferreira, A.M.M.O. Pisco, M.R. Aires-Barros, Partioning of human antibodies in polyethylene glycol-sodium citrate aqueous two-phase systems, Sep. Purif. Technol., 65 (2009) 14.
• [141] P.A.J. Rosa, A.M. Azevedo, M.R.A. Aires-Barros, Application of central composite design to the optimisation of aqueous two-phase extraction of human antibodies, J. Chromatogr. A., 1141 (2007) 50.
• [142] K. Huse, H.J. Böhme, G.H. Scholz, Purification of antibodies by affinity chromatography, J. Biochem. Biophys. Methods, 51 (2002) 217.