A Direct-Infusion- and HPLC-ESI-Orbitrap-MS Approach for the Characterization of Intact PEGylated Proteins
Ines C. Forstenlehner, Johann Holzmann, Kai Scheffler, Wolfgang Wieder, Hansjörg Toll, and Christian G. Huber

Abstract:
The covalent attachment of PEG (PEGylation) is a well-established approach to improve various protein attributes such as decreased immunogenicity and reduced renal clearance. However their characterization by electrospray ionization-mass spectrometry constitutes a challenge due to the overlapping protein charge state pattern and PEG polydispersity. This was overcome by the addition of triethylamine which results in charge reduction and thereby resolved protein spectra. Subsequently we transferred this method to Orbitrap mass spectrometry which enabled us to gain isotopic resolved spectra featuring a median mass accuracy below 1.5 ppm. Using rhG-CSF as a model protein the elucidation of protein oxidation and the separation of the oxidation variants was demonstrated.

The open access article can be found  here

Reviewed by Hans Brandstetter