A Direct-Infusion- and HPLC-ESI-Orbitrap-MS Approach for the Characterization of Intact PEGylated Proteins

Abstract

The characterization of proteins modified with poly(ethylene glycol) (PEG), such as recombinant human granulocyte-colony stimulating factor (PEGylated rhG-CSF or pegfilgrastim), by electrospray ionization-mass spectrometry (ESI-MS) constitutes a challenge due to the overlapping protein charge state pattern and PEG polydispersity. In order to minimize spectral overlaps, charge reduction by means of the addition of amine was applied. Method development for direct-infusion measurements, carried out on an ESI-time-of-flight (ESI-TOF) instrument, demonstrated the potential of triethylamine (TEA) for shifting the charge state pattern toward lower-charged species and of formic acid (FA) for causing higher charging. After successful method transfer to the LTQ Orbitrap XL instrument, isotopically resolved mass spectra could be acquired. With a median mass accuracy of 1.26 ppm, a number-average monoisotopic molecular mass of 40074.64 Da was determined for pegfilgrastim. The direct comparison of three Orbitrap mass spectrometers, namely the LTQ Orbitrap XL, the Exactive, and the Q Exactive, demonstrated that online interfacing to high performance liquid chromatography (HPLC) was only feasible with the Q Exactive, which offers adequate spectral quality on a time scale compatible with chromatographic separation (i.e., 0.2 min acquisition time per chromatographic peak). Finally, the applicability of both direct-infusion Orbitrap MS and HPLC interfaced to Orbitrap MS was demonstrated for the detection of methionine oxidation in pegfilgrastim. Singly, doubly, and triply oxidized species were readily resolved in the chromatogram, while their oxidation status was easily determined from the mass shifts observed in the deconvoluted mass spectra.

Exposure to positively- and negatively-charged plasma cluster ions impairs IgE-binding capacity of indoor cat and fungal allergens

“…Fel d 1 that was collected after PC-ions or sham treatment was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition and proteins were visualized by silver staining following the manufacturer’s instructions (Cosmo Bio, Tokyo, Japan). Proteins separated on SDS-PAGE were identified by liquid chromatography (LC) connected to a mass spectrometer (MS, LTQ Orbitrap XL, Thermo Fisher Scientific, Rockford, IL, USA) using a HPLC column (Develosil ODS-HG-5, Nomura Chemical, Aichi, Japan) and Xcalibur software v2.0.7 (Thermo Fisher Scientific), as described elsewhere [16]. …”

From Synthesis to Characterization of Site-Selective PEGylated Proteins

“…However, the exact determination of the PEG attachment site(s) continues to be highly challenging, especially in a mixture composed of products with differing degrees of PEGylation (Gerislioglu et al, 2018). On the other hand, ESI-TOF has overcome some disadvantages related to polydispersion and the overlapping protein charge pattern of PEGylated proteins (Forstenlehner et al, 2014). Furthermore, ESI-MS is preferred to MALDI due to automated workflow and reduced sample preparation time (Hutanu, 2014).…”

Maintaining consistent quality and clinical performance of biopharmaceuticals

“…For example, pegfilgrastim, a pegylated biopharmaceutical whose heterogeneity was previously impervious to detailed characterization, can now be directly characterized down to site-specific amino acid modifications and individual peg-extension units (see Figure 1(b)) [57,58]. Further, progress in the ability of mass spectrometers to analyze large intact proteins is increasing the utility of this technology for identifying and monitoring functionally relevant product variants.…”