SialEXO® in a Glycoform Analysis of Intact Erythropoietin

January 19, 2022 | References |

In this study, scientists at Leiden University Medical Center present a MALDI FT-ICR MS method for the glycosylation profiling of intact erythropoietin (EPO). The sialidase mix SialEXO significantly reduced the mass spectrum complexity and facilitated the interpretation of the complex spectra of intact EPO.
 

Recombinant human erythropoietin (EPO) is a complex glycoprotein and a successful biopharmaceutical for patients with chronic kidney disease and anemia. The protein carries three N- and one O-glycosylation site. Since the glycosylation profile affects the safety and efficacy of EPO and is defined as a critical quality attribute (CQA), it must be characterized to ensure the quality of the biopharmaceutical.
 
A combination of mass spectrometry (MS)-based methods are typically performed for the in-depth glycosylation analysis of complex glycoproteins, and information obtained from multiple analysis levels needs to be integrated for a comprehensive characterization of EPO glycosylation.
 
In this study, scientists at Leiden University Medical Center present a MALDI FT-ICR MS method for the glycosylation profiling of intact EPO. Three EPO variants were characterized in-depth, and robust and detailed glycosylation profiles were obtained directly from the evaluation of doubly charged EPO glycoforms, without any deconvolution of the mass spectra.
 
The glycosylation profiles obtained by MALDI FT-ICR MS were complex, and by first desialylating the protein using the sialidase mix SialEXO, the mass spectrum complexity was significantly reduced. The MS data obtained from desialylated EPO was then used to determine the “core” glycan structures underneath the heterogenic sialylation as well as identify other modifications potentially leading to isobaric structures. This helped with interpretation of the complex spectra of intact EPO and increased confidence in the peak annotations. Key glycosylation features such as sialylation, acetylation and N-acetyllactosamine repeats were evaluated and found to agree with previously reported data.
 
To conclude, the developed method allowed for a comparison of EPO variants with the potential to be used throughout the manufacturing process, including for clonal selection, batch-to-batch comparison, and biosimilars evaluation.

 


Reference

Lippold et al., 2021. Glycoform analysis of intact erythropoietin by MALDI FT-ICR mass spectrometry. Analytica Chimica Acta. https://doi.org/10.1016/j.aca.2021.339084.

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SialEXO® – Hydrolysis of sialic acids