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In this study, we applied a combination of techniques, including post\u2010column addition of triethylamine in reversed\u2010phase liquid chromatography\u2013mass spectrometry (RPLC\u2010MS) to determine the intact molecular mass, and in\u2010source fragmentation (ISF) and higher\u2010energy collision dissociation\u2013tandem mass spectrometry (HCD\u2010MS\/MS) to identify the PEGylation site. Our results show that both the pegfilgrastim biosimilar candidate and Neulasta lots are mono\u2010PEGylated at the N\u2010terminal end. Furthermore, we show that the combined ISF and HCD\u2010MS\/MS method can be used for identifying the PEGylation sites in the diPEGylated variant of pegfilgrastim. The diPEGylated variant has modification sites at the N\u2010terminal end and a lysine at position 35 in the protein sequence.<\/jats:p>","DOI":"10.1002\/jms.5017","type":"journal-article","created":{"date-parts":[[2024,3,22]],"date-time":"2024-03-22T08:10:46Z","timestamp":1711095046000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Characterization of PEGylation sites in Neulasta and a biosimilar candidate with a combined fragmentation strategy in mass spectrometry analysis"],"prefix":"10.1002","volume":"59","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-9074-2830","authenticated-orcid":false,"given":"Navin","family":"Rauniyar","sequence":"first","affiliation":[{"name":"Tanvex BioPharma USA, Inc.  San Diego California USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alyssa J.","family":"Togle","sequence":"additional","affiliation":[{"name":"Tanvex BioPharma USA, Inc.  San Diego California USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rachel A.","family":"Ronci","sequence":"additional","affiliation":[{"name":"Tanvex BioPharma USA, Inc.  San Diego California USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Diego","family":"Reyes","sequence":"additional","affiliation":[{"name":"Tanvex BioPharma USA, Inc.  San Diego California USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xuemei","family":"Han","sequence":"additional","affiliation":[{"name":"Tanvex BioPharma USA, Inc.  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