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.
摘要：
在为Neulasta开发生物类似产品的过程中，确定完整的分子质量和确认精确的聚乙二醇化位点是至关重要的。在这项研究中,我们应用了多种技术，包括在反相液相色谱-质谱(RPLC-MS)中加入三乙胺以确定完整的分子质量,和源内片段化（ISF）和高能碰撞解离串联质谱（HCD-MS/MS）来识别聚乙二醇化位点。我们的结果表明，pegfilgrastim生物相似物候选物和Neulasta批次在N末端都是单PEG化的。此外，我们表明，ISF和HCD-MS/MS组合方法可用于鉴定pegfilgrastim的二聚乙二醇化变体中的聚乙二醇化位点。二聚乙二醇化变体在蛋白质序列中的N-末端具有修饰位点和位置35处的赖氨酸。
