Abstract
Erythropoietin (EPO) is a heavily glycosylated hormone whose recombinant forms are used for treatment of anaemia. EPO glycosylation is important for its pharmacological properties. An analytical workflow, which can determine EPO glycosylation in an accurate and high-throughput fashion from cell culture supernatant (CCS) in approximately 24 h, offers the possibility to follow changes during production. To address this challenge, we present a complete workflow consisting of protein purification, glycan release, sialic acid derivatization, solid phase extraction, matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) analysis and MassyTools data processing. EPO purification from CCS by anti-EPO antibody coupled Sepharose beads yielded excellent purity with acceptable recovery and was free of glycoform bias. Glycosylation profiles obtained by MALDI-MS were highly comparable to those obtained with an established capillary gel electrophoresis–laser induced fluorescence method. Our method delivers accurate results for the analysis of changes of important glycosylation parameters, such as sialylation and number of N-acetyllactosamine units, for the time course of a fermentation. We could resolve differences in glycosylation between several CCS samples.
Highlights
Erythropoietin (EPO) is a glycoprotein hormone best known for its role in the production of red blood cells, though it has multiple other physiological functions[1]
Distinction of N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc), the number of N-acetyllactosamine (LacNAc) repeats and the degree of O-acetylation (Ac) of sialic acids are relevant for pharmacokinetics[4]
Though some reports have introduced reducing end derivatization in order to allow additional analysis by liquid chromatography with fluorescence detection (LC-FLD)[17, 18], MALDI-MS analysis of non-derivatized glycans omits the intrinsic bias and reduced accuracy associated with this additional sample preparation step[19, 20]
Summary
Erythropoietin (EPO) is a glycoprotein hormone best known for its role in the production of red blood cells, though it has multiple other physiological functions[1]. Four forms, namely epoetin alfa, epoetin beta, epoetin delta and darbepoetin alfa, are distinguished mainly by their glycosylation (for example, in darbepoetin alfa, five amino acids are changed to generate two additional glycosylation sites)[4,5,6] This and the fact that approximately half of EPO molecular weight is due to the glycan content, underlines the importance of glycan analysis for therapeutic EPO. Derivatization of sialic acids can be useful to neutralize the charge and reduce the known instability of sialic acid containing glycans, observed in the reflectron mode of time-of-flight mass detection[21] This phenomenon is often circumvented by desialylation prior to MALDI-MS and parallel analysis of sialylation by complementary techniques[17]. All MALDI-MS methods for EPO glycosylation analysis described in literature report quite extensive sample preparation protocols, often featuring separate steps for desalting, protein removal, removal of excess label and even fractionation either by anion exchange or normal phase (liquid chromatography) LC7, 17–19
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