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

IF 1.9 3区 化学 Q3 BIOCHEMICAL RESEARCH METHODS
Navin Rauniyar, Alyssa J. Togle, Rachel A. Ronci, Diego Reyes, Xuemei Han
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引用次数: 0

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.

利用质谱分析中的组合碎片策略确定 Neulasta 和一种候选生物仿制药中 PEG 化位点的特征。
在开发 Neulasta 的生物类似产品时,必须确定其完整的分子质量并确认精确的 PEG 化位点。在本研究中,我们综合应用了多种技术,包括在反相液相色谱-质谱联用仪(RPLC-MS)中柱后添加三乙胺以确定完整分子质量,以及源内碎片分析(ISF)和高能碰撞解离-串联质谱联用仪(HCD-MS/MS)以确定 PEG 化位点。我们的研究结果表明,pegfilgrastim 候选生物仿制药和 Neulasta 批次在 N 端均为单 PEG 化。此外,我们还发现 ISF 和 HCD-MS/MS 组合方法可用于鉴定 pegfilgrastim 二 PEG 化变体中的 PEG 化位点。双 PEG 化变体的 N 端有修饰位点,蛋白质序列的第 35 位有一个赖氨酸。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Mass Spectrometry
Journal of Mass Spectrometry 化学-光谱学
CiteScore
5.10
自引率
0.00%
发文量
84
审稿时长
1.5 months
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.
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