期刊
ANALYTICA CHIMICA ACTA
卷 1004, 期 -, 页码 58-66出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.aca.2017.12.009
关键词
Multidimensional mass spectrometry; PEGylation; Site-specific characterization; Reversed-phase chromatography; Ion mobility; In-source dissociation
资金
- National Science Foundation [CHE-1308307]
- Direct For Mathematical & Physical Scien [1308307] Funding Source: National Science Foundation
Conjugation of poly(ethylene glycol) (PEG) to protein drugs (PEGylation) is increasingly utilized in the biotherapeutics field because it improves significantly the drugs' circulatory half-life, solubility, and shelf-life. The activity of a PEGylated drug depends on the number, size, and location of the attached PEG chain(s). This study introduces a 2D separation approach, including reversed-phase ultra-performance liquid chromatography (RP-UPLC) and ion mobility mass spectrometry (IM-MS), in order to determine the structural properties of the conjugates, as demonstrated for a PEGylated insulin sample that was prepared by random amine PEGylation. The UPLC dimension allowed separation based on polarity. Electrospray ionization (ESI) of the eluates followed by in-source dissociation (ISD) truncated the PEG chains and created insulin fragments that provided site-specific information based on whether they contained a marker at the potential conjugation sites. Separation of the latter fragments by size and charge in the orthogonal IM dimension (pseudo-4D UPLC-ISD-IM-MS approach) enabled clear detection and identification of the positional isomers formed upon PEGylation. The results showed a highly heterogeneous mixture of singly and multiply conjugated isomers plus unconjugated material. PEGylation was observed on all three possible attachment sites (epsilon-NH2 of LysB29, A-and B-chain N-termini). Each PEGylation site was validated by analysis of the same product after disulfide bond cleavage, so that the PEGylated A-and B-chain could be individually characterized with the same pseudo-4D UPLC-ISD-IM-MS method. (c) 2018 Elsevier B.V. All rights reserved.
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