Subunit mass analysis for monitoring antibody oxidation. Issue 3 (3rd April 2017)

• Record Type: Journal Article
• Authors: Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J.; Hu, Ping
• Is Part Of: MAbs. Volume 9:Issue 3(2017)
• Page Start: 498
• Page End: 505
• Publication Date: 2017-04-03
• Subjects: Fc -- LC-MS -- mass spectrometry -- methionine oxidation -- monoclonal antibody -- posttranslational modifications -- process monitoring -- product characterization -- subunit mass analysis; Monoclonal antibodies -- Therapeutic use -- Periodicals; Monoclonal antibodies -- Periodicals; Antibodies, Monoclonal -- Periodicals; 616.0798
• DOI: 10.1080/19420862.2017.1279773
• Languages: English
• ISSNs: 1942-0862

Abstract

ABSTRACT: Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd' and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation.