Expanding the structural resolution of glycosylation microheterogeneity in therapeutic proteins by salt-free hydrophilic interaction liquid chromatography tandem mass spectrometry
Glycosylation affects the safety and efficacy of therapeutic proteins and is often considered a critical quality attribute (CQA). Therefore, it is important to identify and quantify glycans during drug development. Glycosylation is a highly complex post-translational modification (PTM) due to its st...
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Taylor & Francis Group
2024-12-01
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| Series: | mAbs |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2024.2395503 |
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| author | Yutian Gan Steffen Lippold John Stobaugh Christian Schöneich Feng Yang |
| author_facet | Yutian Gan Steffen Lippold John Stobaugh Christian Schöneich Feng Yang |
| author_sort | Yutian Gan |
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| description | Glycosylation affects the safety and efficacy of therapeutic proteins and is often considered a critical quality attribute (CQA). Therefore, it is important to identify and quantify glycans during drug development. Glycosylation is a highly complex post-translational modification (PTM) due to its structural heterogeneity, i.e. glycosylation site occupancy, glycan compositions, modifications, and isomers. Current analytical tools compromise either structural resolution or site specificity. Hydrophilic interaction liquid chromatography-fluorescence-mass spectrometry (HILIC-FLR-MS) is the gold standard for structural analysis of released glycans, but lacks information on site specificity and occupation. However, HILIC-FLR-MS often uses salt in the solvent, which impairs analysis robustness and sensitivity. Site-specific glycosylation analysis via glycopeptides, upon proteolytic digestion, is commonly performed by reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), but provides only compositional and limited structural glycan information. In this study, we introduce a salt-free, glycopeptide-based HILIC-tandem mass spectrometry (HILIC-MS/MS) method that provides glycan identification, glycan isomer separation and site-specific information simultaneously. Moreover, HILIC-MS/MS demonstrated comparable relative quantification results as released glycan HILIC-FLR-MS. Further, our new method improves the retention of hydrophilic peptides, allowing simultaneous analysis of important CQAs such as deamidation in antibodies. The developed method offers a valuable tool to streamline the site-specific glycosylation analysis of glycoproteins, which is particularly important for the expanding landscape of novel therapeutic formats in the biopharmaceutical industry. |
| format | Article |
| id | doaj-art-c50565fa46114d0ab1802b3e7a995331 |
| institution | Kabale University |
| issn | 1942-0862 1942-0870 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
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| series | mAbs |
| spelling | doaj-art-c50565fa46114d0ab1802b3e7a9953312025-01-31T04:19:38ZengTaylor & Francis GroupmAbs1942-08621942-08702024-12-0116110.1080/19420862.2024.2395503Expanding the structural resolution of glycosylation microheterogeneity in therapeutic proteins by salt-free hydrophilic interaction liquid chromatography tandem mass spectrometryYutian Gan0Steffen Lippold1John Stobaugh2Christian Schöneich3Feng Yang4Protein Analytical Chemistry, Genentech, A Member of the Roche Group, South San Francisco, CA, USAProtein Analytical Chemistry, Genentech, A Member of the Roche Group, South San Francisco, CA, USADepartment of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USADepartment of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USAProtein Analytical Chemistry, Genentech, A Member of the Roche Group, South San Francisco, CA, USAGlycosylation affects the safety and efficacy of therapeutic proteins and is often considered a critical quality attribute (CQA). Therefore, it is important to identify and quantify glycans during drug development. Glycosylation is a highly complex post-translational modification (PTM) due to its structural heterogeneity, i.e. glycosylation site occupancy, glycan compositions, modifications, and isomers. Current analytical tools compromise either structural resolution or site specificity. Hydrophilic interaction liquid chromatography-fluorescence-mass spectrometry (HILIC-FLR-MS) is the gold standard for structural analysis of released glycans, but lacks information on site specificity and occupation. However, HILIC-FLR-MS often uses salt in the solvent, which impairs analysis robustness and sensitivity. Site-specific glycosylation analysis via glycopeptides, upon proteolytic digestion, is commonly performed by reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), but provides only compositional and limited structural glycan information. In this study, we introduce a salt-free, glycopeptide-based HILIC-tandem mass spectrometry (HILIC-MS/MS) method that provides glycan identification, glycan isomer separation and site-specific information simultaneously. Moreover, HILIC-MS/MS demonstrated comparable relative quantification results as released glycan HILIC-FLR-MS. Further, our new method improves the retention of hydrophilic peptides, allowing simultaneous analysis of important CQAs such as deamidation in antibodies. The developed method offers a valuable tool to streamline the site-specific glycosylation analysis of glycoproteins, which is particularly important for the expanding landscape of novel therapeutic formats in the biopharmaceutical industry.https://www.tandfonline.com/doi/10.1080/19420862.2024.2395503Critical quality attributeglycopeptidehydrophilic interaction liquid chromatographymass spectrometry |
| spellingShingle | Yutian Gan Steffen Lippold John Stobaugh Christian Schöneich Feng Yang Expanding the structural resolution of glycosylation microheterogeneity in therapeutic proteins by salt-free hydrophilic interaction liquid chromatography tandem mass spectrometry mAbs Critical quality attribute glycopeptide hydrophilic interaction liquid chromatography mass spectrometry |
| title | Expanding the structural resolution of glycosylation microheterogeneity in therapeutic proteins by salt-free hydrophilic interaction liquid chromatography tandem mass spectrometry |
| title_full | Expanding the structural resolution of glycosylation microheterogeneity in therapeutic proteins by salt-free hydrophilic interaction liquid chromatography tandem mass spectrometry |
| title_fullStr | Expanding the structural resolution of glycosylation microheterogeneity in therapeutic proteins by salt-free hydrophilic interaction liquid chromatography tandem mass spectrometry |
| title_full_unstemmed | Expanding the structural resolution of glycosylation microheterogeneity in therapeutic proteins by salt-free hydrophilic interaction liquid chromatography tandem mass spectrometry |
| title_short | Expanding the structural resolution of glycosylation microheterogeneity in therapeutic proteins by salt-free hydrophilic interaction liquid chromatography tandem mass spectrometry |
| title_sort | expanding the structural resolution of glycosylation microheterogeneity in therapeutic proteins by salt free hydrophilic interaction liquid chromatography tandem mass spectrometry |
| topic | Critical quality attribute glycopeptide hydrophilic interaction liquid chromatography mass spectrometry |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2024.2395503 |
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