Site-Specific Glycosylation Profiling of Protein Subunit and Inactivated Virus Vaccines

Published

July 22, 2026

Author(s)

Zachary Goecker, Meghan Burke Harris, Yi Liu, Yuri Mirokhin, Sergey Sheetlin, Guanghui Wang, Dmitrii Tchekhovskoi, Xiaoyu Yang, Stephen Stein

Abstract

Background/Objectives: Glycosylation can affect vaccine antigen structure and function, making site-specific glycan characterization relevant to antigen quality and comparability. However, quantitative approaches for comparing glycan microheterogeneity remain limited. This study evaluated the utility of the glycopeptide abundance distribution spectra framework for measuring similarity among site-specific glycosylation profiles in vaccines and antigen reference reagents across manufacturing conditions. Methods: Intact N-linked glycopeptides were characterized by nanoflow liquid chromatography–tandem mass spectrometry with stepped-energy fragmentation. Products included monovalent and quadrivalent influenza antigens produced in embryonated eggs, Madin–Darby canine kidney cells, or Spodoptera frugiperda cells, together with a SARS-CoV-2 spike vaccine produced in Spodoptera frugiperda cells and a Chinese hamster ovary cell-produced varicella-zoster virus glycoprotein E vaccine. Site-specific glycan distributions were represented as distribution spectra and compared using NIST MS Search software. Dot-product scores ranging from 0 to 999 quantified similarity. Results: Across measured glycosylation sites, distributions clustered into six recurrent classes. Similarity was high for replicate analyses, conserved influenza components across annual formulations, and matched components from different suppliers within the same production platform (similarity scores = 978, 961, and 960, respectively). Similarity was lower between sites within the same protein, between influenza strains, and between production sources (similarity scores = 554, 540, and 209, respectively). Among production-source comparisons, egg- and Madin–Darby canine kidney-derived profiles were most similar, and the overall ordering of glycosylation similarity was consistent with broad phylogenetic relatedness among production hosts. Conclusions: Distribution spectra-based similarity scoring of vaccine glycoproteins provides a quantitative, reusable approach for documenting site-specific glycosylation microheterogeneity. Using this method, we can conclude that production source is the dominant contributor to variation, whereas replicates, annual formulations, and suppliers within the same production platform are highly consistent.

Citation

Vaccines

Volume

14

Issue

7

Pub Type

Journals

Download Paper

https://doi.org/10.3390/vaccines14070644

Local Download

Keywords

vaccine comparability, subunit vaccine, inactivated vaccine, glycosylation, site-specific, mass spectrometry, proteomics

Citation

Goecker, Z. , Burke Harris, M. , Liu, Y. , Mirokhin, Y. , Sheetlin, S. , Wang, G. , Tchekhovskoi, D. , Yang, X. and Stein, S. (2026), Site-Specific Glycosylation Profiling of Protein Subunit and Inactivated Virus Vaccines, Vaccines, [online], https://doi.org/10.3390/vaccines14070644, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=962109 (Accessed August 1, 2026)

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