title: Characterization of IgG1 immunoglobulins and peptide-Fc fusion proteins by limited proteolysis in conjunction with LC-MS.
abstract: A combinatory approach for the characterization of post-translational and chemical modifications in high molecular weight therapeutic proteins like antibodies and peptide-Fc fusion proteins (MW > or = 50 000 Da) is presented. In this approach, well-established techniques such as limited proteolysis, reversed-phase (RP) high-performance liquid chromatography (HPLC), and in-line mass spectrometry (MS) were combined for the characterization of a monoclonal IgG1 antibody and three different peptide-Fc fusion proteins. The one commonality of these molecules is the presence of a similarly accessible lysine residue either located in the flexible hinge region of the antibody or in the flexible linker of the peptide-Fc fusion proteins. Applying limited proteolysis using endoproteinase Lys-C resulted in the predominant cleavage C-terminal of this lysine residue. The created fragments, two identical Fab domain fragments and one Fc domain fragment derived from the IgG1 antibody and one Fc domain fragment and each of the three individual peptide moieties generated from the peptide-Fc fusion proteins, were readily accessible for complete separation by RP-HPLC and detailed characterization by in-line MS analysis. This approach facilitated rapid detection of a variety of chemical modifications such as methionine oxidation, disulfide bond scrambling, and reduction as well as the characterization of various carbohydrate chains. We found limited proteolysis followed by RP-HPLC-MS to be less time-consuming for sample preparation, analysis, and data interpretation than traditional peptide mapping procedures. At the same time, the reduced sample complexity provided superior chromatographic and mass spectral resolution than the analysis of the corresponding intact molecules or a large number of enzymatically generated fragments.
authors.affiliation: Department of Pharmaceutics, Amgen Incorporated, One Amgen Center Drive, Thousand Oaks, California 91320, USA. gerdk@amgen.com
authors.forename: Gerd R
authors.initials: GR
authors.lastname: Kleemann
authors.forename: Jill
authors.initials: J
authors.lastname: Beierle
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Nichols
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Dillon
authors.forename: Gary D
authors.initials: GD
authors.lastname: Pipes
authors.forename: Pavel V
authors.initials: PV
authors.lastname: Bondarenko
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins