title: Structural characterization of expressed monoclonal antibodies by single sample mass spectral analysis after IdeS proteolysis.
abstract: Simple and rapid methods for analysis of monoclonal antibody structure and post-translational modifications are increasingly needed due to the explosion of therapeutic monoclonal antibodies and monoclonal antibody applications. Mass spectral analysis is a powerful method for characterizing monoclonal antibodies. Recent discovery and commercialization of the Immunoglobulin G-degrading enzyme of Streptococcus pyogene (IdeS protease) has facilitated and improved the generation of antibody fragments of suitable size to allow characterization of the structure of the entire antibody molecule via analysis of just a few fragments. In this study, we coupled IdeS fragmentation and simultaneous reduction and alkylation of the resultant fragments using tributylphosphine and iodoacetamide to prepare samples in about 2 h. Following simple introduction of a single, unseparated mixture of alkylated fragments into a mass spectrometer, detailed structural information is obtained, covering the entire antibody molecule. The large majority of the glycoforms present on the single, conserved N-linked glycosylation site of the heavy chain is elucidated, although some of the very low abundance glycoforms are not determined by this protocol. The ease, simplicity, speed, and power of this method make it attractive for analysis of the developmental stages and production batches of therapeutic monoclonal antibodies.
authors.affiliation: Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0575, USA. Electronic address: terry.kirley@uc.edu.
authors.forename: Terence L
authors.initials: TL
authors.lastname: Kirley
authors.affiliation: Department of Cancer Biology, Proteomics Laboratory, College of Medicine, University of Cincinnati, 3125 Eden Avenue, Cincinnati, OH 45267-0521, USA.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Greis
authors.affiliation: Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0575, USA.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Norman
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001426:Bacterial Proteins / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D011487:Protein Conformation; D059748:Proteolysis
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; C442815:Mac-1-like protein, Streptococcus
keywords: Glycoform determination; IdeS cleavage; Mass spectrometry; Monoclonal antibody; Post-translational modifications; Tributylphosphine reduction