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Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry

Glycosylation is a critical quality attribute of monoclonal antibody (mAb) therapeutics. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) is an invaluable technology for the characterization of protein glycosylation. HILIC/MS-based glycan analysis relies on the library sear...

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Autores principales: Singh, Sumit K., Lee, Kelvin H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8786911/
https://www.ncbi.nlm.nih.gov/pubmed/35087805
http://dx.doi.org/10.3389/fbioe.2021.805788
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author Singh, Sumit K.
Lee, Kelvin H.
author_facet Singh, Sumit K.
Lee, Kelvin H.
author_sort Singh, Sumit K.
collection PubMed
description Glycosylation is a critical quality attribute of monoclonal antibody (mAb) therapeutics. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) is an invaluable technology for the characterization of protein glycosylation. HILIC/MS-based glycan analysis relies on the library search using Glucose Units (GU) and accurate mass (AM) as the primary search parameters for identification. However, GU-based identifications are gradient-dependent and are not suitable for applications where separation gradients need to be optimized to analyze complex samples or achieve higher throughput. Additionally, the workflow requires calibration curves (using dextran ladder) to be generated for each analysis campaign, which in turn, are used to derive the GU values of the separated glycan species. To overcome this limitation, we employed a two-step strategy for targeted glycan analysis of a mAb expressed in Chinese Hamster Ovary (CHO) cells. The first step is to create a custom library of the glycans of interest independent of GU values (thereby eliminating the need for a calibration curve) and instead uses AM and retention time (RT) as the primary search variables. The second step is to perform targeted glycan screening using the custom-built library. The developed workflow was applied for targeted glycan analysis of a mAb expressed in CHO for 1) cell line selection 2) characterizing the day-wise glycan evolution in a model mAb during a fed-batch culture, 3) assessing the impact of different media conditions on glycosylation, and 4) evaluating the impact of two different process conditions on glycosylation changes in a model mAb grown in a bioreactor. Taken together, the data presented in this study provides insights into the sources of glycan heterogeneity in a model mAb that are seen during its commercial manufacturing.
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spelling pubmed-87869112022-01-26 Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry Singh, Sumit K. Lee, Kelvin H. Front Bioeng Biotechnol Bioengineering and Biotechnology Glycosylation is a critical quality attribute of monoclonal antibody (mAb) therapeutics. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) is an invaluable technology for the characterization of protein glycosylation. HILIC/MS-based glycan analysis relies on the library search using Glucose Units (GU) and accurate mass (AM) as the primary search parameters for identification. However, GU-based identifications are gradient-dependent and are not suitable for applications where separation gradients need to be optimized to analyze complex samples or achieve higher throughput. Additionally, the workflow requires calibration curves (using dextran ladder) to be generated for each analysis campaign, which in turn, are used to derive the GU values of the separated glycan species. To overcome this limitation, we employed a two-step strategy for targeted glycan analysis of a mAb expressed in Chinese Hamster Ovary (CHO) cells. The first step is to create a custom library of the glycans of interest independent of GU values (thereby eliminating the need for a calibration curve) and instead uses AM and retention time (RT) as the primary search variables. The second step is to perform targeted glycan screening using the custom-built library. The developed workflow was applied for targeted glycan analysis of a mAb expressed in CHO for 1) cell line selection 2) characterizing the day-wise glycan evolution in a model mAb during a fed-batch culture, 3) assessing the impact of different media conditions on glycosylation, and 4) evaluating the impact of two different process conditions on glycosylation changes in a model mAb grown in a bioreactor. Taken together, the data presented in this study provides insights into the sources of glycan heterogeneity in a model mAb that are seen during its commercial manufacturing. Frontiers Media S.A. 2022-01-11 /pmc/articles/PMC8786911/ /pubmed/35087805 http://dx.doi.org/10.3389/fbioe.2021.805788 Text en Copyright © 2022 Singh and Lee. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Singh, Sumit K.
Lee, Kelvin H.
Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry
title Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry
title_full Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry
title_fullStr Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry
title_full_unstemmed Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry
title_short Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry
title_sort characterization of monoclonal antibody glycan heterogeneity using hydrophilic interaction liquid chromatography-mass spectrometry
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8786911/
https://www.ncbi.nlm.nih.gov/pubmed/35087805
http://dx.doi.org/10.3389/fbioe.2021.805788
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