Immunoglobulin specificity of TG19318: a novel synthetic ligand for antibody affinity purification

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  • Abbreviations used:ELISA, enzyme-linked immunosorbent assay; FDA, Food and Drugs Administration; IgG, immunoglobulin G; IgA, immunoglobulin A; IgE, immunoglobulin E; IgM, immunoglobulin M; MAb, monoclonal antibody; PAM, protein A mimetic; RP-HPLC, reverse phase high-performance liquid affinity chromatography; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis

Abstract

A synthetic ligand [TG19318], able to mimic protein A in the recognition of the immunoglobulin Fc portion, has been previously identified in our laboratory through the synthesis and screening of multimeric combinatorial peptide libraries. In this study we have fully characterized its applicability in affinity chromatography for the downstream processing of antibodies, examining the specificity and selectivity for polyclonal and monoclonal immunoglobulins derived from different sources. Ligand specificity was broader than protein A, since IgG deriving from human, cow, horse, pig, mouse, rat, rabbit, goat and sheep sera, IgY obtained from egg yolk, and IgM, IgA and IgE were efficiently purified on TG19318 affinity columns. Adsorbed antibodies were conveniently eluted by a buffer change to 0.1 M acetic acid or 0.1 M sodium bicarbonate pH 9, with full retention of immunological properties. Monoclonal antibodies deriving from cell culture supernatants or ascitic fluids were also conveniently purified on TG19318 affinity columns, even from very diluted samples. The affinity constant for the TG19318–IgG interaction was 0.3 µM, as determined by optical biosensor measurements. Under optimized conditions, antibody purity after affinity purification was close to 95%, as determined by densitometric scanning of SDS–PAGE gels of purified fractions, and maximal column capacity reached 25 mg Ig/ml support. In vivo toxicity studies in mice indicated a ligand oral toxicity greater than 2000 mg kg−1 while intravenous toxicity was close to 150 mg kg−1. Validation of antibody affinity purification processes for therapeutic use, a very complex, laborious and costly procedure, is going to be simplified by the use of TG19318, which could reduce considerably the presence of biological contaminants in the purified preparation, a very recurrent problem when using recombinant or extractive biomolecules as affinity ligands. Copyright © 1998 John Wiley & Sons, Ltd.

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