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Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping
The evaluation of the protein glycosylation states of samples of aflibercept obtained from three different regions was conducted by site-specific N-linked glycan microheterogeneity profiling. Glycopeptide-based nano-LC MSMS mapping of tryptic-digested samples of each aflibercept lot provided site-sp...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9569749/ https://www.ncbi.nlm.nih.gov/pubmed/36233110 http://dx.doi.org/10.3390/ijms231911807 |
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author | Lee, Ju Yeon Choi, Jin-Woong Hwang, Seoyoung Hahm, Sung Ho Ahn, Yeong Hee |
author_facet | Lee, Ju Yeon Choi, Jin-Woong Hwang, Seoyoung Hahm, Sung Ho Ahn, Yeong Hee |
author_sort | Lee, Ju Yeon |
collection | PubMed |
description | The evaluation of the protein glycosylation states of samples of aflibercept obtained from three different regions was conducted by site-specific N-linked glycan microheterogeneity profiling. Glycopeptide-based nano-LC MSMS mapping of tryptic-digested samples of each aflibercept lot provided site-specific information about glycan microheterogeneity on each of the five N-glycosylation sites (two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region). Next, the glycopeptide-mapping results obtained from the three different aflibercept lots were compared to evaluate the similarity between the samples. Three aflibercept lots showed a high degree of similarity in glycan composition, fucosylation level, sialylation level, and branching, when all five N-glycosylation sites were assessed together as a group. On the other hand, noticeable variations between lots in the glycan types and sialylation levels on the two sites of the VEGFR-2 domain were observed when each of the five N-glycosylation sites were assessed using the glycopeptide-based method. The presence of N-glycolylneuraminic acid (NeuGc) glycans, which may mediate adverse immune reactions in antibody therapeutics, were also detected on the sites of VEGFR1 and VEGFR2 domains, but not on the IgG Fc domain site. These results imply that analyses of the glycosylation profiles of fusion proteins containing multiple N-glycosylation sites, such as aflibercept, being done as a part of quality control for the therapeutics manufacturing process or for biosimilar development, can be done with a more applicable outcome by assessing each site separately. |
format | Online Article Text |
id | pubmed-9569749 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-95697492022-10-17 Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping Lee, Ju Yeon Choi, Jin-Woong Hwang, Seoyoung Hahm, Sung Ho Ahn, Yeong Hee Int J Mol Sci Article The evaluation of the protein glycosylation states of samples of aflibercept obtained from three different regions was conducted by site-specific N-linked glycan microheterogeneity profiling. Glycopeptide-based nano-LC MSMS mapping of tryptic-digested samples of each aflibercept lot provided site-specific information about glycan microheterogeneity on each of the five N-glycosylation sites (two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region). Next, the glycopeptide-mapping results obtained from the three different aflibercept lots were compared to evaluate the similarity between the samples. Three aflibercept lots showed a high degree of similarity in glycan composition, fucosylation level, sialylation level, and branching, when all five N-glycosylation sites were assessed together as a group. On the other hand, noticeable variations between lots in the glycan types and sialylation levels on the two sites of the VEGFR-2 domain were observed when each of the five N-glycosylation sites were assessed using the glycopeptide-based method. The presence of N-glycolylneuraminic acid (NeuGc) glycans, which may mediate adverse immune reactions in antibody therapeutics, were also detected on the sites of VEGFR1 and VEGFR2 domains, but not on the IgG Fc domain site. These results imply that analyses of the glycosylation profiles of fusion proteins containing multiple N-glycosylation sites, such as aflibercept, being done as a part of quality control for the therapeutics manufacturing process or for biosimilar development, can be done with a more applicable outcome by assessing each site separately. MDPI 2022-10-05 /pmc/articles/PMC9569749/ /pubmed/36233110 http://dx.doi.org/10.3390/ijms231911807 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Lee, Ju Yeon Choi, Jin-Woong Hwang, Seoyoung Hahm, Sung Ho Ahn, Yeong Hee Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping |
title | Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping |
title_full | Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping |
title_fullStr | Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping |
title_full_unstemmed | Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping |
title_short | Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping |
title_sort | site-specific glycan microheterogeneity evaluation of aflibercept fusion protein by glycopeptide-based lc-msms mapping |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9569749/ https://www.ncbi.nlm.nih.gov/pubmed/36233110 http://dx.doi.org/10.3390/ijms231911807 |
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