Navin Rauniyar, Alyssa J. Togle, Rachel A. Ronci, Diego Reyes, Xuemei Han
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Characterization of PEGylation sites in Neulasta and a biosimilar candidate with a combined fragmentation strategy in mass spectrometry analysis
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.
期刊介绍:
The Journal of Mass Spectrometry publishes papers on a broad range of topics of interest to scientists working in both fundamental and applied areas involving the study of gaseous ions.
The aim of JMS is to serve the scientific community with information provided and arranged to help senior investigators to better stay abreast of new discoveries and studies in their own field, to make them aware of events and developments in associated fields, and to provide students and newcomers the basic tools with which to learn fundamental and applied aspects of mass spectrometry.