Discrepancies between High-Resolution Native and Glycopeptide-Centric Mass Spectrometric Approaches: A Case Study into the Glycosylation of Erythropoietin Variants.
Tomislav ČavalAlexander BuettnerMarkus HabergerDietmar ReuschAlbert J R HeckPublished in: Journal of the American Society for Mass Spectrometry (2021)
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.
Keyphrases
- mass spectrometry
- liquid chromatography
- high resolution
- tandem mass spectrometry
- gas chromatography
- high resolution mass spectrometry
- high performance liquid chromatography
- capillary electrophoresis
- copy number
- simultaneous determination
- recombinant human
- high speed
- dna methylation
- healthcare
- regulatory t cells
- big data
- optical coherence tomography
- single cell
- smoking cessation