title: Detection of IgG aggregation by a high throughput method based on extrinsic fluorescence.
abstract: The utility of extrinsic fluorescence as a tool for high throughput detection of monoclonal antibody aggregates was explored. Several IgG molecules were thermally stressed and the high molecular weight species were fractionated using size-exclusion chromatography (SEC). The isolated aggregates and monomers were studied by following the fluorescence of an extrinsic probe, SYPRO Orange. The dye displayed high sensitivity to structurally altered, aggregated IgG structures compared to the native form, which resulted in very low fluorescence in the presence of the dye. An example of the application is presented here to demonstrate the properties of this detection method. The fluorescence assay was shown to correlate with the SEC method in quantifying IgG aggregates. The fluorescent probe method appears to have potential to detect protein particles that could not be analyzed by SEC. This method may become a powerful high throughput tool to detect IgG aggregates in pharmaceutical solutions and to study other protein properties involving aggregation. It can also be used to study the kinetics of antibody particle formation, and perhaps allow identification of the species, which are the early building blocks of protein particles.
authors.affiliation: Formulation and Analytical Resources, AW2/D3152, Amgen, Inc., 1201 Amgen Court West, Seattle, Washington, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: He
authors.forename: Duke H
authors.initials: DH
authors.lastname: Phan
authors.forename: Sabine
authors.initials: S
authors.lastname: Hogan
authors.forename: Robert
authors.initials: R
authors.lastname: Bailey
authors.forename: Gerald W
authors.initials: GW
authors.lastname: Becker
authors.forename: Linda O
authors.initials: LO
authors.lastname: Narhi
authors.forename: Vladimir I
authors.initials: VI
authors.lastname: Razinkov
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry*; D002850:Chromatography, Gel; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D046911:Macromolecular Substances / Q000032:analysis; D019999:Pharmaceutical Solutions / Q000032:analysis; D011506:Proteins / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D019999:Pharmaceutical Solutions; D011506:Proteins