Phospho-l-tyrosine-agarose chromatography: Adsorption of human IgG and its proteolytic fragments

Author(s)

Pavan, Gisele Luiza ; Lazzarotto Bresolin, Igor Tadeu ; Velho Muzio, Aline Ferreira ; Cunha, Daniele Celestino ; Alves Bueno, Sonia Maria

• Document type: Journal article
• Source: BIOMEDICAL CHROMATOGRAPHY; v. 33, n. 1, p. 11-pg., 2019-01-01.

Abstract

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. (AU)

• FAPESP's process: 13/12705-9 - O-phospho-L-tyrosine (OPT): new ligand of mixed-mode chromatography for purification of polyclonal human IgG
• Grantee: Sonia Maria Alves Bueno
• Support Opportunities: Regular Research Grants