title: Understanding the conformational impact of chemical modifications on monoclonal antibodies with diverse sequence variation using hydrogen/deuterium exchange mass spectrometry and structural modeling.
abstract: Chemical modifications can potentially induce conformational changes near the modification site and thereby impact the safety and efficacy of protein therapeutics. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a powerful analytical technique with high spatial resolution and sensitivity in detecting such local conformational changes. In this study, we utilized HDX-MS combined with structural modeling to examine the conformational impact on monoclonal antibodies (mAbs) caused by common chemical modifications including methionine (Met) oxidation, aspartic acid (Asp) isomerization, and asparagine (Asn) deamidation. Four mAbs with diverse sequences and glycosylation states were selected. The data suggested that the impact of Met oxidation was highly dependent on its location and glycosylation state. For mAbs with normal glycosylation in the Fc region, oxidation of the two conserved Met252 and Met428 (Kabat numbering) disrupted the interface interactions between the CH2 and CH3 domains, thus leading to a significant decrease in CH2 domain thermal stability as well as a slight increase in aggregation propensity. In contrast, Met oxidation in the variable region and CH3 domain had no detectable impact on mAb conformation. For aglycosylated mAb, Met oxidation could cause a more global conformational change to the whole CH2 domain, coincident with the larger decrease in thermal stability and significant increase in aggregation rate. Unlike Met oxidation, Asn deamidation and Asp isomerization mostly had very limited effects on mAb conformation, with the exception of succiminide intermediate formation which induced a measurable local conformational change to be more solvent protected. Structural modeling suggested that the succinimide intermediate was stabilized by adjacent aromatic amino acids through ring-ring stacking interactions.
authors.affiliation: Biopharmaceutical Analytical Sciences, Biopharm R&D, GlaxoSmithKline , 709 Swedeland Road, King of Prussia, Pennsylvania 19406, United States.
authors.forename: Aming
authors.initials: A
authors.lastname: Zhang
authors.forename: Ping
authors.initials: P
authors.lastname: Hu
authors.forename: Paul
authors.initials: P
authors.lastname: MacGregor
authors.forename: Yu
authors.initials: Y
authors.lastname: Xue
authors.forename: Haihong
authors.initials: H
authors.lastname: Fan
authors.forename: Peter
authors.initials: P
authors.lastname: Suchecki
authors.forename: Leonard
authors.initials: L
authors.lastname: Olszewski
authors.forename: Aston
authors.initials: A
authors.lastname: Liu
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D003903:Deuterium; D006031:Glycosylation; D006859:Hydrogen; D013058:Mass Spectrometry / Q000379:methods*; D010084:Oxidation-Reduction; D011487:Protein Conformation
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D006859:Hydrogen; D003903:Deuterium