TY - JOUR
T1 - Purification of human serum immunoglobulins using immobilized metal affinity chromatography with ethylenediamine triacetic acid as chelating agent
AU - González-Ortega, Omar
AU - Guzmán, Roberto
N1 - Publisher Copyright:
© Taylor & Francis Group, LLC.
Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.
PY - 2015/1/2
Y1 - 2015/1/2
N2 - Purified immunoglobulins are required for a wide range of applications in biotechnology such as laboratory and clinical reagents, novel therapeutics, and immunoaffinity ligands in chromatography. The present work describes the effective separation of immunoglobulins from mixtures of synthetic immunoglobulin solutions and human serum using immobilized metal affinity chromatography and specified pH and imidazole gradients. Ethylenediamine triacetic acid was used as the chelating agent and Cu(II) as the immobilized ion on an agarose matrix. The modified agarose gel presented Cu(II) and immunoglobulin capacities of 65 μmol/mL and 46 mg/mL, respectively. pH and imidazole gradients were performed in order to find the best conditions for the affinity of immunoglobulins toward the immobilized Cu(II) ions. The chelating systems were able to effectively retain immunoglobulins and showed no measurable affinity towards human serum albumin under all working conditions. In the purification of immunoglobulins from human serum, a pH gradient was able to release immunoglobulins (1.3 mg) with a total purity of 60% (and a purity of 84% for 65% recovery of all adsorbed protein), while an imidazole gradient allowed the recovery of 0.8 mg of immunoglobulins with a total purity of 75% (and a purity of 83% for 70% recovery of all adsorbed protein).
AB - Purified immunoglobulins are required for a wide range of applications in biotechnology such as laboratory and clinical reagents, novel therapeutics, and immunoaffinity ligands in chromatography. The present work describes the effective separation of immunoglobulins from mixtures of synthetic immunoglobulin solutions and human serum using immobilized metal affinity chromatography and specified pH and imidazole gradients. Ethylenediamine triacetic acid was used as the chelating agent and Cu(II) as the immobilized ion on an agarose matrix. The modified agarose gel presented Cu(II) and immunoglobulin capacities of 65 μmol/mL and 46 mg/mL, respectively. pH and imidazole gradients were performed in order to find the best conditions for the affinity of immunoglobulins toward the immobilized Cu(II) ions. The chelating systems were able to effectively retain immunoglobulins and showed no measurable affinity towards human serum albumin under all working conditions. In the purification of immunoglobulins from human serum, a pH gradient was able to release immunoglobulins (1.3 mg) with a total purity of 60% (and a purity of 84% for 65% recovery of all adsorbed protein), while an imidazole gradient allowed the recovery of 0.8 mg of immunoglobulins with a total purity of 75% (and a purity of 83% for 70% recovery of all adsorbed protein).
KW - IMAC
KW - chelating agent
KW - ethylenediamine triacetic acid
KW - immunoglobulins purification
KW - protein adsorption
KW - protein purification
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U2 - 10.1080/10826076.2014.883534
DO - 10.1080/10826076.2014.883534
M3 - Article
AN - SCOPUS:84907682535
VL - 38
SP - 74
EP - 81
JO - Journal of Liquid Chromatography and Related Technologies
JF - Journal of Liquid Chromatography and Related Technologies
SN - 1082-6076
IS - 1
ER -