Characterization of PEGylation sites in Neulasta and a biosimilar candidate with a combined fragmentation strategy in mass spectrometry analysis

Abstract In the development of biosimilar products to Neulasta, it is essential to determine the intact molecular mass and confirm precise PEGylation sites. In this study, we applied a combination of techniques, including post‐column addition of triethylamine in reversed‐phase liquid chromatography–mass spectrometry (RPLC‐MS) to determine the intact molecular mass, and in‐source fragmentation (ISF) and higher‐energy collision dissociation–tandem mass spectrometry (HCD‐MS/MS) to identify the PEGylation site. Our results show that both the pegfilgrastim biosimilar candidate and Neulasta lots are mono‐PEGylated at the N‐terminal end. Furthermore, we show that the combined ISF and HCD‐MS/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‐terminal end and a lysine at position 35 in the protein sequence.