title: Using lysine-reactive fluorescent dye for surface characterization of a mAb.
abstract: The biopharmaceutical industry increasingly demands thorough characterization of protein conformation and conformational dynamics to ensure product quality and consistency. Here, we present a chromatography-based method that is able to characterize protein conformation and conformational dynamics at peptide level resolution in a high-throughput manner. The surface lysine residues of the protein were labeled with a fluorescent dye prior to enzyme digestion. The resulting peptide maps were monitored by fluorescence detection where fluorescence peak area indicates higher solvent accessibility at a specific site. The peptides of reactivity difference and the extent of the difference can be detected by HPLC with fluorescent detector alone, whereas the identity of these peptides can then be determined by mass spectrometry if desired. We first demonstrated this method is suitable for probing protein surface/conformation by studying the effect of deglycosylation on a recombinant mAb, IgG 1. We then applied our method to study the interaction of the mAb with a common excipient, polysorbate-20 (PS-20). The presence of PS-20 increased the fluorescent labeling of several lysine residues on the mAb. This result provides a first insight into PS20-mAb interaction at peptide level resolution.
authors.affiliation: Protein Analytical Chemistry, Genentech, Inc, South San Francisco, California, 94080.
authors.forename: Ming
authors.initials: M
authors.lastname: Lei
authors.forename: Yung-Hsiang
authors.initials: YH
authors.lastname: Kao
authors.forename: Christian
authors.initials: C
authors.lastname: Schöneich
mesh_terms: D000085:Acetates / Q000737:chemistry* / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002867:Chromones / Q000737:chemistry* / Q000378:metabolism; D005079:Excipients / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D057166:High-Throughput Screening Assays; D008239:Lysine; D008958:Models, Molecular; D010449:Peptide Mapping; D011136:Polysorbates / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013499:Surface Properties; D013678:Technology, Pharmaceutical / Q000379:methods
chemical_list: D000085:Acetates; C400304:Alexa 350; D000911:Antibodies, Monoclonal; D002867:Chromones; D005079:Excipients; D005456:Fluorescent Dyes; D011136:Polysorbates; D008239:Lysine
keywords: LC-MS; chromatography; protein formulation; protein structure; surfactants