title: A Quantitative Microtiter Assay for Sialylated Glycoform Analyses Using Lectin Complexes.
abstract: Fidelity of glycan structures is a key requirement for biotherapeutics, with carbohydrates playing an important role for therapeutic efficacy. Comprehensive glycan profiling techniques such as liquid chromatography (LC) and mass spectrometry (MS), while providing detailed description of glycan structures, require glycan cleavage, labeling, and paradigms to deconvolute the considerable data sets they generate. On the other hand, lectins as probes on microarrays have recently been used in orthogonal approaches for in situ glycoprofiling but require analyte labeling to take advantage of the capabilities of automated microarray readers and data analysis they afford. Herein, we describe a lectin-based microtiter assay (lectin-enzyme-linked immunosorbent assay [ELISA]) to quantify terminal glycan moieties, applicable to in vitro and in-cell glycan-engineered Fc proteins as well as intact IgGs from intravenous immunoglobulin (IVIG), a blood product containing pooled polyvalent IgG antibodies extracted from plasma from healthy human donors. We corroborate our findings with industry-standard LC-MS profiling. This "customizable" ELISA juxtaposes readouts from multiple lectins, focusing on a subset of glycoforms, and provides the ability to discern single- versus dual-arm glycosylation while defining levels of epitopes at sensitivities comparable to MS. Extendable to other biologics, this ELISA can be used stand-alone or complementary to MS for quantitative glycan analysis.
authors: Momenta Pharmaceuticals, Inc., Cambridge, MA, USA.
authors: Karunya
authors: K
authors: Srinivasan
authors: Momenta Pharmaceuticals, Inc., Cambridge, MA, USA sroy@momentapharma.com.
authors: Sucharita
authors: S
authors: Roy
authors: Momenta Pharmaceuticals, Inc., Cambridge, MA, USA.
authors: Nathaniel
authors: N
authors: Washburn
authors: Momenta Pharmaceuticals, Inc., Cambridge, MA, USA.
authors: Sandra F
authors: SF
authors: Sipsey
authors: Momenta Pharmaceuticals, Inc., Cambridge, MA, USA.
authors: Robin
authors: R
authors: Meccariello
authors: Momenta Pharmaceuticals, Inc., Cambridge, MA, USA.
authors: James W
authors: JW
authors: Meador
authors: Momenta Pharmaceuticals, Inc., Cambridge, MA, USA.
authors: Leona E
authors: LE
authors: Ling
authors: Momenta Pharmaceuticals, Inc., Cambridge, MA, USA.
authors: Anthony M
authors: AM
authors: Manning
authors: Momenta Pharmaceuticals, Inc., Cambridge, MA, USA.
authors: Ganesh V
authors: GV
authors: Kaundinya
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D016756:Immunoglobulins, Intravenous / Q000378:metabolism; D037102:Lectins / Q000378:metabolism*; D013058:Mass Spectrometry; D011134:Polysaccharides / Q000378:metabolism
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D016756:Immunoglobulins, Intravenous; D037102:Lectins; D011134:Polysaccharides
keywords: ELISA; IVIG; glycoform; lectin; sialylation