Therapeutic proteins and peptides are commonly modified with the addition of polyethylene glycol (PEG) residues. The process is known as PEGylation and overcomes drug instability, inhibits rapid clearance from circulation by the kidneys or proteolytic breakdown, and decreases immune system activation to reduce antibody formation. As therapeutic modalities, however, these PEGylated proteins are subject to stringent quality control and pharmaceutical regulation. Before approval, PEGylated proteins must be fully characterized in terms of sequence, PEGylation site and number of residues, and degradation pathways. Mass spectrometry (MS) can be used to reliably characterize PEGylated proteins.
Forstenlehner et al. (2014) have optimized an MS approach, developing the protocol from its original matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) approach to electrospray ionization−time of flight (ESI-TOF).1 They further refined their method, transferring it to an LTQ Orbitrap XL Hybrid Ion Trap-Orbitrap mass spectrometer (Thermo Scientific) before running comparisons on two newer-generation Orbitrap MS instruments, the Exactive Orbitrap mass spectrometer and the Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (both Thermo Scientific). This approach was counter to previous wisdom, which stated that Orbitrap MS was not appropriate for measuring PEGylated proteins because ionized products would be held up by the inline C-trap unit.
Previously, to overcome MS problems such as polydispersity and overlapping protein charge-state patterns that complicate the reading of mass spectra, researchers would de-PEGylate the samples prior to analysis. Although that technique was successful in preparing the samples for MS analysis, features such as PEGylation site and length were lost. Following up on previous research, Forstenlehner and colleagues investigated different sample preparation methods to reduce charge, shift the charge-state pattern and improve MS resolution by reducing spectral overlap.
The researchers chose recombinant human granulocyte colony stimulating factor (rhG-CSF, or pegfilgastrim)—an extensively characterized PEGylated therapeutic protein used to ameliorate neutropenia associated with chemotherapy during cancer treatment—for their experimental protocols. They evaluated organic acids and solvents such as methanol, acetonitrile and isopropanol as solvents for preparing samples prior to initial ESI-TOF MS. Methanol was the best solvent for the rhG-CSF standards in terms of spectral data clarity; however, the clearest results were obtained using triethylamine (TEA) to shift the charge-state pattern into the best mass range, thus improving resolution in the Orbitrap-based instruments. Additional refining using formic acid at 30 mmol L-1 in addition to TEA at 10 mmol L-1 produced the best signal-to-noise ratios within the mass range and m/z cutoff at 4000.
Once validated, the investigators optimized the method for analysis with the LTQ Orbitrap XL mass spectrometer. After instrument calibration to improve spectral quality, the researchers used deconvolution algorithms to investigate isotope peak spacing and quantify PEG variants. Although deconvolution did reveal separate isotope peaks, Forstenlehner et al. found that data points were missed and that some variants, therefore, were not exposed.
Use of the validated method with two newer-generation Orbitrap-based instruments proved more successful. The researchers obtained higher quality scans in less time and with greater resolution, commenting that improved hardware and processing contributed to the enhanced results. For example, the Q Exactive mass spectrometer detected 79 peaks over 0.5 minutes, whereas the LTQ Orbitrap XL mass spectrometer took 30 minutes to read this number.
Finally, the researchers used direct-infusion methodology with the Q Exactive mass spectrometer to examine chemical oxidation of methionine residues on the rhG-CSF standards, as a measure of deterioration was expected with the drug. Although MS analysis could not pinpoint the exact methionine location, it did give an approximation of the degree to which the sample was oxidized. Coupling the Q Exactive mass spectrometer with ion-pair reversed-phase high-performance liquid chromatography (HPLC—using the UltiMate 3000 system from Thermo Scientific) gave similar results for oxidative products.
By demonstrating full characterization of the various PEG products arising from pegfilgastrim, the authors are confident that their sample preparation protocol and use of the Q Exactive mass spectrometer fully optimizes the characterization of PEGylated therapeutic proteins.
1. Forstenlehner, I.C., et al. (2014) “A Direct-Infusion- and HPLC-ESI-Orbitrap-MS Approach for the Characterization of Intact PEGylated Proteins,” Analytical Chemistry, 86 (pp. 826−34), dx.doi.org/10.1021/ac403390y.
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