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

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 lot s 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 i n the protein sequence.

Source: Journal of Mass Spectrometry - April 2, 2024 Category: Chemistry Authors: Navin Rauniyar, Alyssa J. Togle, Rachel A. Ronci, Diego Reyes, Xuemei Han Tags: RESEARCH ARTICLE Source Type: research