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Phospho‐ l ‐tyrosine‐agarose chromatography: Adsorption of human IgG and its proteolytic fragments
Author(s) -
Pavan Gisele Luiza,
Bresolin Igor Tadeu Lazzarotto,
Muzio Aline Ferreira Velho,
Cunha Daniele Celestino,
Bueno Sonia Maria Alves
Publication year - 2019
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.4397
Subject(s) - chemistry , chromatography , agarose , adsorption , tris , elution , bovine serum albumin , immunoglobulin g , affinity chromatography , antibody , biochemistry , organic chemistry , enzyme , immunology , biology
The behavior of human immunoglobulin G (IgG) and antigen‐binding fragment (Fab fragment) adsorption onto phospho‐ l ‐tyrosine immobilized on agarose (P‐Tyr‐agarose) was evaluated by pseudoaffinity chromatography. The effects of buffer systems MES, MOPS, Bis–Tris, Tris–HCl and sodium phosphate (NaP) and pH on IgG adsorption were studied and high purity values were obtained (96%, based on ELISA analysis of albumin, transferrin and immunoglobulins A, G and M) when IgG was purified from human plasma diluted in 10 mmol L −1 NaP buffer at pH 6.0. The capture of IgG by the P‐Tyr‐agarose was also promising, since 91% of the IgG was adsorbed when plasma was diluted in 25 mmol L −1 MES buffer at pH 5.5, recommending its use for IgG depletion from human plasma under this condition. The experimental data on IgG adsorption kinetics were in agreement with the pseudo‐second‐order model. The adsorption isotherm data were well described by the Langmuir–Freundlich model with the value of parameter n being <1 (0.72), indicating negative cooperativity. Selectivity was achieved on P‐Tyr‐agarose from digested human IgG in HEPES 25 mmol L −1 buffer at pH 7.0 where Fab fragments were obtained in eluted fractions without Fc fragments (but with uncleaved IgG) with 86.2% recovery.