Evaluation of immobilized metal-ion affinity chromatography (IMAC) as a technique for IgG(1) monoclonal antibodies purification: the effect of chelating ligand and support.
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 IgG(1) 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 IgG(1) using PEVA-CM-Asp-Zn(II). The anti-TNP MAb could be eluted under mild pH conditions causing no loss of antigen binding capacity.
Bresolin
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IT
School of Chemical Engineering, University of Campinas, UNICAMP, P.O. Box 6066, 13083-970 Campinas, SP, Brazil.
Borsoi-Ribeiro
M
M
Tamashiro
W M S C
WM
Augusto
E F P
EF
Vijayalakshmi
M A
MA
Bueno
S M A
SM
D000327:Adsorption; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D001224:Aspartic Acid / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D005029:Ethylenediamines / Q000737:chemistry; D007098:Imino Acids / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification*; D008024:Ligands; D008567:Membranes, Artificial; D008670:Metals / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D013499:Surface Properties; D015032:Zinc / Q000737:chemistry
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D005029:Ethylenediamines; D007098:Imino Acids; D007074:Immunoglobulin G; D008024:Ligands; D008567:Membranes, Artificial; D008670:Metals; D001224:Aspartic Acid; D009532:Nickel; D015032:Zinc; D009571:Nitrilotriacetic Acid; C008109:iminodiacetic acid