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Evaluation of Immobilized Metal-Ion Affinity Chromatography (IMAC) as a Technique for IgG1 Monoclonal Antibodies Purification: The Effect of Chelating Ligand and Support

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Abstract

Monoclonal antibodies (MAbs) have been used for therapies and some analytical procedures as highly purified molecules. Many techniques have been applied and studied, focusing on monoclonal antibodies purification. In this study, an immobilized metal affinity chromatography membrane was developed and evaluated for the purification of anti-TNP IgG1 mouse MAbs from cell culture supernatant after precipitation with a 50% saturated ammonium sulfate solution. The chelating ligands iminodiacetic acid, carboxymethylated aspartic acid (CM-Asp), nitrilotriacetic acid, and tris (carboxymethyl) ethylenediamine in agarose gels with immobilized Ni(II) and Zn(II) ions were compared for the adsorption and desorption of MAbs. The most promising chelating ligand—CM-Asp—was then coupled to poly(ethylene vinyl alcohol) (PEVA) hollow fiber membranes. According to SDS-PAGE and ELISA analyses, a higher selectivity and a purification factor of 85.9 (fraction eluted at 500 mM Tris) were obtained for IgG1 using PEVA-CM-Asp-Zn(II). The anti-TNP MAb could be eluted under mild pH conditions causing no loss of antigen binding capacity.

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Acknowledgments

The authors gratefully acknowledge the financial support of FAPESP—Fundação de Amparo à Pesquisa do Estado de São Paulo—SP, Brazil and CNPq—Conselho Nacional de Desenvolvimento Científico e Tecnológico—Brasília, DF, Brazil.

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Authors and Affiliations

  1. School of Chemical Engineering, University of Campinas, UNICAMP, P.O. Box 6066, 13083-970, Campinas, SP, Brazil

    I. T. L. Bresolin, M. Borsoi-Ribeiro & S. M. A. Bueno

  2. Institute of Biology, University of Campinas, UNICAMP, P.O. Box 6109, 13083-970, Campinas, SP, Brazil

    W. M. S. C. Tamashiro

  3. Institute for Technological Research of the State of São Paulo, Av. Prof. Almeida Prado, 532, 05508-901, Sao Paulo, SP, Brazil

    E. F. P. Augusto

  4. Centre for Bioseparation Technology, VIT University Vellore, Vellore 632 014, Tamil Nadu, India

    M. A. Vijayalakshmi

Authors
  1. I. T. L. Bresolin
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  2. M. Borsoi-Ribeiro
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  3. W. M. S. C. Tamashiro
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  4. E. F. P. Augusto
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  5. M. A. Vijayalakshmi
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  6. S. M. A. Bueno
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Corresponding author

Correspondence to S. M. A. Bueno.

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Bresolin, I.T.L., Borsoi-Ribeiro, M., Tamashiro, W.M.S.C. et al. Evaluation of Immobilized Metal-Ion Affinity Chromatography (IMAC) as a Technique for IgG1 Monoclonal Antibodies Purification: The Effect of Chelating Ligand and Support. Appl Biochem Biotechnol 160, 2148–2165 (2010). https://doi.org/10.1007/s12010-009-8734-5

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  • DOI: https://doi.org/10.1007/s12010-009-8734-5

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