title: A lectin-based gold nanoparticle assay for probing glycosylation of glycoproteins.
abstract: We report a glycoanalysis method in which lectins are used to probe the glycans of therapeutic glycoproteins that are adsorbed on gold nanoparticles. A model mannose-presenting glycoprotein, ribonuclease B (RNase B), and the therapeutic monoclonal antibody (mAb) rituximab, were found to adsorb spontaneously and non-specifically to bare gold nanoparticles such that glycans were accessible for lectin binding. Addition of a multivalent binding lectin, such as concanavalin A (Con A), to a solution of the modified gold nanoparticles resulted in cross-linking of the nanoparticles. This phenomenon was evidenced within 1 min by a change in the hydrodynamic diameter, D(H), measured by dynamic light scattering (DLS) and a shift and increase in absorbance of the plasmon resonance band of the gold nanoparticles. By combining the sugar-binding specificity and the cross-linking capabilities of lectins, the non-specific adsorption of glycoproteins to gold surfaces, and the unique optical reporting properties of gold nanoparticles, a glycosylation pattern of rituximab could be generated. This assay provides advantages over currently used glycoanalysis methods in terms of short analysis time, simplicity of the conjugation method, convenience of simple spectroscopic detection, and feasibility of providing glycan characterization of the protein drug product by using a variety of binding lectins.
authors.affiliation: National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA.
authors.forename: Germarie
authors.initials: G
authors.lastname: Sánchez-Pomales
authors.forename: Todd A
authors.initials: TA
authors.lastname: Morris
authors.forename: James B
authors.initials: JB
authors.lastname: Falabella
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Tarlov
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Zangmeister
mesh_terms: D000327:Adsorption; D000911:Antibodies, Monoclonal / Q000737:chemistry; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry; D001709:Biotechnology / Q000379:methods; D005428:Flocculation Tests; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006046:Gold / Q000737:chemistry*; D008027:Light; D053768:Metal Nanoparticles / Q000737:chemistry*; D008956:Models, Chemical; D037121:Plant Lectins / Q000737:chemistry* / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry; D000069283:Rituximab; D012542:Scattering, Radiation; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D006023:Glycoproteins; D037121:Plant Lectins; D000069283:Rituximab; D006046:Gold; D012260:Ribonucleases; C029600:ribonuclease B