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Discrepancies between High-Resolution Native and Glycopeptide-Centric Mass Spectrometric Approaches: A Case Study into the Glycosylation of Erythropoietin Variants.
- Source :
-
Journal of the American Society for Mass Spectrometry [J Am Soc Mass Spectrom] 2021 Aug 04; Vol. 32 (8), pp. 2099-2104. Date of Electronic Publication: 2021 Apr 15. - Publication Year :
- 2021
-
Abstract
- Glycosylation represents a critical quality attribute modulating a myriad of physiochemical properties and effector functions of biotherapeutics. Furthermore, a rising landscape of glycosylated biotherapeutics including biosimilars, biobetters, and fusion proteins harboring complicated and dynamic glycosylation profiles requires tailored analytical approaches capable of characterizing their heterogeneous nature. In this work, we perform in-depth evaluation of the glycosylation profiles of three glycoengineered variants of the widely used biotherapeutic erythropoietin. We analyzed these samples in parallel using a glycopeptide-centric liquid chromatography/mass spectrometry approach and high-resolution native mass spectrometry. Although for all of the studied variants the glycopeptide and native mass spectrometry data were in good qualitative agreement, we observed substantial quantitative differences arising from ionization deficiencies and unwanted neutral losses, in particular, for sialylated glycopeptides in the glycoproteomics approach. However, the latter provides direct information about glycosite localization. We conclude that the combined parallel use of native mass spectrometry and bottom-up glycoproteomics offers superior characterization of glycosylated biotherapeutics and thus provides a valuable attribute in the characterization of glycoengineered proteins and other complex biotherapeutics.
- Subjects :
- Biosimilar Pharmaceuticals chemistry
Chromatography, Liquid
Erythropoietin chemistry
Erythropoietin genetics
Glycopeptides chemistry
Glycosylation
Proteomics methods
Biosimilar Pharmaceuticals analysis
Erythropoietin analysis
Erythropoietin metabolism
Glycopeptides analysis
Mass Spectrometry methods
Subjects
Details
- Language :
- English
- ISSN :
- 1879-1123
- Volume :
- 32
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- Journal of the American Society for Mass Spectrometry
- Publication Type :
- Academic Journal
- Accession number :
- 33856811
- Full Text :
- https://doi.org/10.1021/jasms.1c00060