Investigation of Site-Specific Differences in Glycan Microheterogeneity by N-Glycopeptide Mapping of VEGFR-IgG Fusion Protein

Hahm, Young Hye; Lee, Ju Yeon; Ahn, Yeong Hee

doi:10.3390/molecules24213924

Cited by 7 publications

(5 citation statements)

References 32 publications

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“…It is comprised of vascular endothelial growth factor receptors 1 and 2 (VEGFR-1, VEGFR-2) fused to the Fc domain of human IgG1, and contains a total of five N-glycosylation sites (two sites from the VEGFR-1 region, two sites from the VEGFR-2 region, and one site from the human IgG Fc region). While most therapeutic antibodies contain a single N-glycosylation site on the Fc portion of the protein, aflibercept, which contains four extra N-glycosylation sites on its VEGF receptor domains, exhibits different glycosylation profiles among glycosylation sites [ 19 ]. Consequently, a method of analysis is required that is capable of differentiating the glycan microheterogeneity site-specifically of the multiply glycosylated fusion protein.…”

Section: Introductionmentioning

confidence: 99%

“…A glycopeptide-based mapping method using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) can be a powerful alternative for the glycosite-specific characterization of complex glycoproteins containing multiple N-glycosylation sites [ 19 , 20 , 21 , 22 ]. In this study, we used LC-ESI MS/MS for glycopeptide-based mapping of the aflibercept fusion protein.…”

Section: Introductionmentioning

confidence: 99%

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Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping

Lee

Choi²,

Hwang³

et al. 2022

IJMS

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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.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping

Lee

Choi²,

Hwang³

et al. 2022

IJMS

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“…High amounts of mannosidic structures (>50%) were reported on other Fc fusions expressed in plants [ 43 ] and might be the consequence of insufficient secretion. Such structures were detected on CHO-produced aflibercept, albeit to a much lesser extent [ 11 , 44 ]. Our results show that mannosidic structures of aflibercept do not impact on VEGF binding; however, some recombinant fusion proteins containing high mannose-type glycans exhibit reduced serum half-life [ 45 ].…”

Section: Discussionmentioning

confidence: 99%

Efficient Expression of Functionally Active Aflibercept with Designed N-glycans

Keshvari,

Melnik,

Sun

et al. 2024

Antibodies

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Aflibercept is a therapeutic recombinant fusion protein comprising extracellular domains of human vascular endothelial growth factor receptors (VEGFRs) and IgG1-Fc. It is a highly glycosylated protein with five N-glycosylation sites that might impact it structurally and/or functionally. Aflibercept is produced in mammalian cells and exhibits large glycan heterogeneity, which hampers glycan-associated investigations. Here, we report the expression of aflibercept in a plant-based system with targeted N-glycosylation profiles. Nicotiana benthamiana-based glycoengineering resulted in the production of aflibercept variants carrying designed carbohydrates, namely, N-glycans with terminal GlcNAc and sialic acid residues, herein referred to as AFLIGnGn and AFLISia, respectively. Both variants were transiently expressed in unusually high amounts (2 g/kg fresh leaf material) in leaves and properly assembled to dimers. Mass spectrometric site-specific glycosylation analyses of purified aflibercept showed the presence of two to four glycoforms in a consistent manner. We also demonstrate incomplete occupancy of some glycosites. Both AFLIGnGn and AFLISia displayed similar binding potency to VEGF165, with a tendency of lower binding to variants with increased sialylation. Collectively, we show the expression of functionally active aflibercept in significant amounts with controlled glycosylation. The results provide the basis for further studies in order to generate optimized products in the best-case scenario.

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“…Several glycoforms using hybrid high-performance liquid chromatography-MS approaches [159], such as 24 glycoengineered erythropoietin variants with varying glycan branching and sialylation levels, are crucial parameters for biotherapeutic efficacy.…”

Section: Glycoformsmentioning

confidence: 99%

A Critical Analysis of the FDA’s Omics-Driven Pharmacodynamic Biomarkers to Establish Biosimilarity

Niazi

2023

Pharmaceuticals

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Demonstrating biosimilarity entails comprehensive analytical assessment, clinical pharmacology profiling, and efficacy testing in patients for at least one medical indication, as required by the U.S. Biologics Price Competition and Innovation Act (BPCIA). The efficacy testing can be waived if the drug has known pharmacodynamic (PD) markers, leaving most therapeutic proteins out of this concession. To overcome this, the FDA suggests that biosimilar developers discover PD biomarkers using omics technologies such as proteomics, glycomics, transcriptomics, genomics, epigenomics, and metabolomics. This approach is redundant since the mode-action-action biomarkers of approved therapeutic proteins are already available, as compiled in this paper for the first time. Other potential biomarkers are receptor binding and pharmacokinetic profiling, which can be made more relevant to ensure biosimilarity without requiring biosimilar developers to conduct extensive research, for which they are rarely qualified.

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Investigation of Site-Specific Differences in Glycan Microheterogeneity by N-Glycopeptide Mapping of VEGFR-IgG Fusion Protein

Cited by 7 publications

References 32 publications

Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping

Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping

Efficient Expression of Functionally Active Aflibercept with Designed N-glycans

A Critical Analysis of the FDA’s Omics-Driven Pharmacodynamic Biomarkers to Establish Biosimilarity

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