Multi-Attribute Monitoring of Complex Erythropoetin Beta Glycosylation by GluC Liquid Chromatography–Mass Spectrometry Peptide Mapping

Abstract

Recombinant human erythropoetin (EPO) is an important biopharmaceutical mainly used for the treatment of anemia. It is highly heterogeneous because of common amino acid chemical degradations known to occur in protein therapeutics (e.g., oxidation and deamidation) and its complex glycosylation profile. Recently, multi-attribute monitoring (MAM), i.e., the quantification of multiple post-translational and chemical modifications in a single peptide mapping liquid chromatography–mass spectrometry (LC-MS)-based method, has received increased attention for the analysis of antibody-like biotherapeutic proteins. In this study, an MAM method for examination of residue-specific glycan profiles of EPO was established. The MAM method, by virtue of the increased sensitivity and selectivity provided with LC-MS, yielded additional site-specific information not afforded by the conventional quality control (QC) methods. Low abundant glycans as well as additional post-translational and chemical modifications could also be simultaneously detected by the MAM method. Our results demonstrate that desialylated N-oligosaccharides (DeNO) and N-acetylneuraminic acids (Neu5Ac) could be monitored by the developed MAM approach with data readout highly comparable to QC methods, while differences were observed for charge isoform distribution. In summary, the comparative data obtained demonstrate that MAM by LC-MS peptide mapping can, in principle, adequately replace selected QC methods and would add value to the in-process control and release testing strategy of EPO.

Discrepancies between High-Resolution Native and Glycopeptide-Centric Mass Spectrometric Approaches: A Case Study into the Glycosylation of Erythropoietin Variants

“… 22 , 24 Next, EPO-1 glycopeptides were generated by GluC digestion, which enables coverage of all glycosylation sites, followed by liquid chromatography/mass spectrometry (LC-MS) analysis. 25 Using our previously described algorithm, 22 , 26 we used GluC bottom-up data to simulate a native MS spectrum. When we then compared the measured and simulated native MS spectra, we obtained a low similarity score of 0.66 ( Figure 1 C).…”

“… 28 For these reasons, we sought to next characterize an EPO variant exhibiting partial N -glycan occupancy (EPO-2) at the first (N24) glycosylation site as measured by our multiattribute method. 25 We, again, performed native MS analysis of desialylated EPO-2 ( Figure 2 A). We identified 13 glycan compositions where the most abundant one corresponded to EPO-2 carrying four tetra-antennary N -glycans and an O -glycan.…”

“…Samples with modified glycan profile EPO-1 and EPO-2 were obtained as previously described. 25 Briefly, EPO drug substance was separated by reverse-phase high-performance liquid chromatography (RP-HPLC) on a Grace VYDAC C4 column using 0.1% TFA in purified water as solvent A and increasing volumetric ratios of 0.1% TFA in ACN as solvent B. Fractions from early (EPO-1) and late (EPO-2) elution time points were separated, collected, and further purified by a repeated RP-HPLC. Fractions containing EPO-1 and EPO-2 were collected again subjected to anion exchange chromatography at 2 to 8 °C on a eiethylaminoethyl (DEAE) sepharose fast flow anion exchange chromatography resin (GE Healthcare).…”

Cysteine Aminoethylation Enables the Site-Specific Glycosylation Analysis of Recombinant Human Erythropoietin using Trypsin

“…MS2 scans were acquired by CID with a normalized collision energy of 35% for the five most intense parent ions. Detailed information about the MS settings can be found in Information S2 …”

Strategies for Proteome-Wide Quantification of Glycosylation Macro- and Micro-Heterogeneity

“…Glycosylation plays critical roles in protein functions and cellular processes, and its aberration is closely associated with human diseases, including cancers [111], neurodegenerative diseases [112,113], immunology disorders [49] and virus infection [114]. Quantitative glycoproteomics studies characterize disease-related micro-and macro-heterogeneous glycosylation changes in purified proteins (e.g., immunoglobulins and therapeutic proteins) [49,[115][116][117][118][119][120][121][122] and complex samples, including serum [52,66,67], urine [51,123], cerebrospinal fluid [124], milk [125], tissues, cells and exosomes [126,127]. Altered protein glycosylation is a hallmark of various cancers and has become a promising target for disease biomarkers [128].…”