{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,26]],"date-time":"2026-10-26T15:51:43Z","timestamp":1793029903526,"version":"4.5.0"},"reference-count":28,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,3,25]],"date-time":"2026-03-25T00:00:00Z","timestamp":1774396800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background\/Objectives: Mass spectrometry-based multi-attribute methods (MAM) have the potential to transform therapeutic protein analysis by enabling comprehensive monitoring of multiple quality attributes in a single assay. However, the widespread adoption of MAM is hindered by significant challenges, most notably artificial oxidation during sample preparation and analysis. This report summarizes long-term operational observations and several case studies that substantiate this concern. Methods: A tryptic digest, high-resolution LC-MS MAM workflow was applied to an Fc-fusion protein and multiple antibody-based therapeutics, with a frozen reference standard analyzed in each run for system suitability and longitudinal trending. Oxidation excursions were investigated by comparing laboratories, consumables, LC-MS configurations, and other method parameters. Results: In a seven-year trending record, apparent total methionine oxidation in the Fc-fusion protein reference standard showed an abrupt, sustained increase (up to ~5-fold); the shift was traced to a specific bag of microcentrifuge-tubes used during buffer exchange and resolved after those tubes were discontinued. In an antibody\u2013drug conjugate, observed methionine oxidation was strongly influenced by the sample preparation procedure. In other antibodies, variability of observed methionine oxidation was attributed to on-column oxidation, which produced a broad and noisy peak that interferes with automated peak integration. EDTA flushing reduced this feature, implicating exposure to metal ions. Conclusions: While advances continue to address many MAM challenges, artificial oxidation remains unpredictable and constitutes a major obstacle to robust implementation in regulated QC environments. Enhanced control strategies and further research are urgently needed to ensure reliable therapeutic protein analysis. Such control strategies include consumable qualification and change control, system suitability\/trending using a reference standard, metal management across LC flow path\/column lifecycle, reduction of trifluoracetic acid (TFA) exposure, data analysis to safeguard excessive on-column oxidation, etc.<\/jats:p>","DOI":"10.3390\/ph19040528","type":"journal-article","created":{"date-parts":[[2026,3,25]],"date-time":"2026-03-25T10:07:13Z","timestamp":1774433233000},"page":"528","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Artificial Oxidation: A Major Challenge in Implementing Multi-Attribute Methods for Therapeutic Protein Analysis"],"prefix":"10.3390","volume":"19","author":[{"given":"Yaokai","family":"Duan","sequence":"first","affiliation":[{"name":"Process Development, Amgen Inc., Thousand Oaks, CA 91320, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Lanzillotti","sequence":"additional","affiliation":[{"name":"Process Development, Amgen Inc., Cambridge, MA 02142, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dylan L.","family":"Riggs","sequence":"additional","affiliation":[{"name":"Process Development, Amgen Inc., Cambridge, MA 02142, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Albana","family":"Nito","sequence":"additional","affiliation":[{"name":"Quality, Amgen Inc., Thousand Oaks, CA 91320, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junnichi","family":"Mijares","sequence":"additional","affiliation":[{"name":"Process Development, Amgen Inc., Thousand Oaks, CA 91320, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-7194-096X","authenticated-orcid":false,"given":"Amanda","family":"Helms","sequence":"additional","affiliation":[{"name":"Process Development, Amgen Inc., Cambridge, MA 02142, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carl","family":"Ly","sequence":"additional","affiliation":[{"name":"Process Development, Amgen Inc., Cambridge, MA 02142, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kevin","family":"Millea","sequence":"additional","affiliation":[{"name":"Process Development, Amgen Inc., Cambridge, MA 02142, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xingwen","family":"Li","sequence":"additional","affiliation":[{"name":"Process Development, Amgen Inc., Cambridge, MA 02142, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Zhang","sequence":"additional","affiliation":[{"name":"Process Development, Amgen Inc., Cambridge, MA 02142, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8326-6714","authenticated-orcid":false,"given":"Zhongqi","family":"Zhang","sequence":"additional","affiliation":[{"name":"Process Development, Amgen Inc., Thousand Oaks, CA 91320, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"8354","DOI":"10.1021\/ac901193n","article-title":"Large-scale identification and quantification of covalent modifications in therapeutic proteins","volume":"81","author":"Zhang","year":"2009","journal-title":"Anal. 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