Abstract 3214: Definition of biosimilars: Energy Resolved Oxonium Ion Monitoring (Erexim) technology grasps detailed N-glycan microheterogeneity on therapeutic antibodies.

Abstract

Abstract The patent expiry of innovative antibody drugs has provoked a great deal of concern over generic drugs. Reduction of medical costs by generics is undoubtedly beneficial to all countries, however, major issues about bioequivalence and immunogenicity remain incompletely addressed. The first draft guidance on the evaluation of biosimilarity was published by FDA in Feb 2012, in which emphasis was placed on the importance of evaluating minor structural differences that can significantly affect the potency and safety of biopharmaceuticals, with specific reference to glycosylation patterns on antibody drugs. We propose here a new method for validating the microheterogeneity of N-glycans on therapeutic antibodies. The continuous collision energy scanning of oligosaccharide-derived fragment ions (oxonium ions) illustrated both comprehensive structural information of glycans on glycopeptides and their quantitative abundance ratio simultaneously in a single analysis of a triple quadrupole mass spectrometer. We showed that oxonium ion m/z 138 could be a specific reporter ion for sensitive quantification of glycopeptides, regardless of glycan structures. Indeed the quantitation limit of glycopeptide was 30 attomole and, importantly, even very rare structural isomers were quantifiable, which existed only 0.1% of all glycoforms. We named this technology Energy Resolved Oxonium Ion Monitoring (Erexim) and utilized for the evaluation of lot-to-lot glycoform variations of monoclonal therapeutic antibodies trastuzumab and bevacizumab. The quantitative profiles of 4 lots (n = 4 each) revealed that N-glycans on both drugs showed gradually shortened tendency with time. Surprisingly some abundant glycoforms on both antibody drugs exhibited 10% changes between two continuous lots. We further applied Erexim technology to in-depth analysis of cetuximab glycosylation pattern, previously known to induce severe anaphylaxis. Our method successfully provided a comprehensive structural view of Fab- and Fc-glycans simultaneously in 30 minutes analysis, which uncovered lots of critical non-human type antigens only on the Fab region of cetuximab, such as Gal(α1-3)Gal or Neu5Gc structures. Hence in the process of future biosimilar approval, the Erexim technology we demonstrated herein has potential to define “how similar it is”. Citation Format: Koji Ueda, Atsuhiko Toyama, Taka-Aki Sato, Yusuke Nakamura, Hidewaki Nakagawa. Definition of biosimilars: Energy Resolved Oxonium Ion Monitoring (Erexim) technology grasps detailed N-glycan microheterogeneity on therapeutic antibodies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3214. doi:10.1158/1538-7445.AM2013-3214 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.