Cargando…

O-Glycoproteomic analysis of engineered heavily glycosylated fusion proteins using nanoHILIC-MS

Recombinant protein engineering design affects therapeutic properties including protein efficacy, safety, and immunogenicity. Importantly, glycosylation modulates glycoprotein therapeutic pharmacokinetics, pharmacodynamics, and effector functions. Furthermore, the development of fusion proteins requ...

Descripción completa

Detalles Bibliográficos
Autores principales: Cavallero, Gustavo J., Wang, Yan, Nwosu, Charles, Gu, Sheng, Meiyappan, Muthuraman, Zaia, Joseph
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9568489/
https://www.ncbi.nlm.nih.gov/pubmed/36136114
http://dx.doi.org/10.1007/s00216-022-04318-7
_version_ 1784809647921692672
author Cavallero, Gustavo J.
Wang, Yan
Nwosu, Charles
Gu, Sheng
Meiyappan, Muthuraman
Zaia, Joseph
author_facet Cavallero, Gustavo J.
Wang, Yan
Nwosu, Charles
Gu, Sheng
Meiyappan, Muthuraman
Zaia, Joseph
author_sort Cavallero, Gustavo J.
collection PubMed
description Recombinant protein engineering design affects therapeutic properties including protein efficacy, safety, and immunogenicity. Importantly, glycosylation modulates glycoprotein therapeutic pharmacokinetics, pharmacodynamics, and effector functions. Furthermore, the development of fusion proteins requires in-depth characterization of the protein integrity and its glycosylation to evaluate their critical quality attributes. Fc-fusion proteins can be modified by complex glycosylation on the active peptide, the fragment crystallizable (Fc) domain, and the linker peptides. Moreover, the type of glycosylation and the glycan distribution at a given glycosite depend on the host cell line and the expression system conditions that significantly impact safety and efficacy. Because of the inherent heterogeneity of glycosylation, it is necessary to assign glycan structural detail for glycoprotein quality control. Using conventional reversed-phase LC–MS methods, the different glycoforms at a given glycosite elute over a narrow retention time window, and glycopeptide ionization is suppressed by co-eluting non-modified peptides. To overcome this drawback, we used nanoHILIC-MS to characterize the complex glycosylation of UTI-Fc, a fusion protein that greatly increases the half-life of ulinastatin. By this methodology, we identified and characterized ulinastatin glycopeptides at the Fc domain and linker peptide. The results described herein demonstrate the advantages of nanoHILIC-MS to elucidate glycan features on glycotherapeutics that fail to be detected using traditional reversed-phase glycoproteomics. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00216-022-04318-7.
format Online
Article
Text
id pubmed-9568489
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Springer Berlin Heidelberg
record_format MEDLINE/PubMed
spelling pubmed-95684892022-10-16 O-Glycoproteomic analysis of engineered heavily glycosylated fusion proteins using nanoHILIC-MS Cavallero, Gustavo J. Wang, Yan Nwosu, Charles Gu, Sheng Meiyappan, Muthuraman Zaia, Joseph Anal Bioanal Chem Research Paper Recombinant protein engineering design affects therapeutic properties including protein efficacy, safety, and immunogenicity. Importantly, glycosylation modulates glycoprotein therapeutic pharmacokinetics, pharmacodynamics, and effector functions. Furthermore, the development of fusion proteins requires in-depth characterization of the protein integrity and its glycosylation to evaluate their critical quality attributes. Fc-fusion proteins can be modified by complex glycosylation on the active peptide, the fragment crystallizable (Fc) domain, and the linker peptides. Moreover, the type of glycosylation and the glycan distribution at a given glycosite depend on the host cell line and the expression system conditions that significantly impact safety and efficacy. Because of the inherent heterogeneity of glycosylation, it is necessary to assign glycan structural detail for glycoprotein quality control. Using conventional reversed-phase LC–MS methods, the different glycoforms at a given glycosite elute over a narrow retention time window, and glycopeptide ionization is suppressed by co-eluting non-modified peptides. To overcome this drawback, we used nanoHILIC-MS to characterize the complex glycosylation of UTI-Fc, a fusion protein that greatly increases the half-life of ulinastatin. By this methodology, we identified and characterized ulinastatin glycopeptides at the Fc domain and linker peptide. The results described herein demonstrate the advantages of nanoHILIC-MS to elucidate glycan features on glycotherapeutics that fail to be detected using traditional reversed-phase glycoproteomics. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00216-022-04318-7. Springer Berlin Heidelberg 2022-09-22 2022 /pmc/articles/PMC9568489/ /pubmed/36136114 http://dx.doi.org/10.1007/s00216-022-04318-7 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Paper
Cavallero, Gustavo J.
Wang, Yan
Nwosu, Charles
Gu, Sheng
Meiyappan, Muthuraman
Zaia, Joseph
O-Glycoproteomic analysis of engineered heavily glycosylated fusion proteins using nanoHILIC-MS
title O-Glycoproteomic analysis of engineered heavily glycosylated fusion proteins using nanoHILIC-MS
title_full O-Glycoproteomic analysis of engineered heavily glycosylated fusion proteins using nanoHILIC-MS
title_fullStr O-Glycoproteomic analysis of engineered heavily glycosylated fusion proteins using nanoHILIC-MS
title_full_unstemmed O-Glycoproteomic analysis of engineered heavily glycosylated fusion proteins using nanoHILIC-MS
title_short O-Glycoproteomic analysis of engineered heavily glycosylated fusion proteins using nanoHILIC-MS
title_sort o-glycoproteomic analysis of engineered heavily glycosylated fusion proteins using nanohilic-ms
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9568489/
https://www.ncbi.nlm.nih.gov/pubmed/36136114
http://dx.doi.org/10.1007/s00216-022-04318-7
work_keys_str_mv AT cavallerogustavoj oglycoproteomicanalysisofengineeredheavilyglycosylatedfusionproteinsusingnanohilicms
AT wangyan oglycoproteomicanalysisofengineeredheavilyglycosylatedfusionproteinsusingnanohilicms
AT nwosucharles oglycoproteomicanalysisofengineeredheavilyglycosylatedfusionproteinsusingnanohilicms
AT gusheng oglycoproteomicanalysisofengineeredheavilyglycosylatedfusionproteinsusingnanohilicms
AT meiyappanmuthuraman oglycoproteomicanalysisofengineeredheavilyglycosylatedfusionproteinsusingnanohilicms
AT zaiajoseph oglycoproteomicanalysisofengineeredheavilyglycosylatedfusionproteinsusingnanohilicms