title: The criticality of high-resolution N-linked carbohydrate assays and detailed characterization of antibody effector function in the context of biosimilar development.
abstract: Accurate measurement and functional characterization of antibody Fc domain N-linked glycans is critical to successful biosimilar development. Here, we describe the application of methods to accurately quantify and characterize the N-linked glycans of 2 IgG1 biosimilars with effector function activity, and show the potential pitfalls of using assays with insufficient resolution. Accurate glycan assessment was combined with glycan enrichment using lectin chromatography or production with glycosylation inhibitors to produce enriched pools of key glycan species for subsequent assessment in cell-based antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity effector function assays. This work highlights the challenges of developing high-quality biosimilar candidates and the need for modern biotechnology capabilities. These results show that high-quality analytics, combined with sensitive cell-based assays to study in vivo mechanisms of action, is an essential part of biosimilar development.
authors.affiliation: a Physical Chemistry Characterization; Sandoz Biopharmaceuticals ; Oberhaching , Germany.
authors.forename: Lowell J
authors.initials: LJ
authors.lastname: Brady
authors.forename: Jyoti
authors.initials: J
authors.lastname: Velayudhan
authors.forename: Devi B
authors.initials: DB
authors.lastname: Visone
authors.forename: Ken C
authors.initials: KC
authors.lastname: Daugherty
authors.forename: Jeff L
authors.initials: JL
authors.lastname: Bartron
authors.forename: Michael
authors.initials: M
authors.lastname: Coon
authors.forename: Cabot
authors.initials: C
authors.lastname: Cornwall
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Hinckley
authors.forename: Lisa
authors.initials: L
authors.lastname: Connell-Crowley
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000920:Antibody-Dependent Cell Cytotoxicity; D001681:Biological Assay; D059451:Biosimilar Pharmaceuticals / Q000737:chemistry*; D002460:Cell Line; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D059451:Biosimilar Pharmaceuticals; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011134:Polysaccharides
keywords: ACN, acetonitrile; ADCC; ADCC, antibody-dependent cell-mediated cytotoxicity; AHGs, afucosylated hybrid glycans; AMBGs, afucosylated monoantennary and biantennary glycans; BGGs, β-galactosylated glycans; CDC; CDC, complement-dependent cytotoxicity; CHO, Chinese hamster ovary; CQA, critical quality attribute; Con A, concanavalin A lectin; EIC, extracted ion current; FT, flow-through; Fc domain; HC, heavy chain; HGs, hybrid glycans; HILIC, hydrophilic interaction liquid chromatography; HMGs, high-mannose glycans; HPLC, high performance liquid chromatography; LC, light chain; MS, mass spectrometry; MS/MS, tandem mass spectrometry; N-linked carbohydrates; PQA, product quality attribute; SGs, sialylated glycans; SM, starting material; TRIS, tris(hydroxymethyl)aminomethane; antibody; biosimilar; effector function; glycosylation; mAb; mAb, monoclonal antibody