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

Hahm Hahm,Ahn Ahn,Lee Lee

doi:10.3390/molecules24213924

Hahm Hahm, Ahn Ahn + Show 1 more

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https://doi.org/10.3390/molecules24213924

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Abstract

A biosimilar fusion protein VEGFR-IgG consisting of vascular endothelial growth factor receptors 1 and 2 (VEGFR-1, VEGFR-2) and the Fc portion of human IgG1 was prepared for this study. The prepared fusion protein was expected to possess a total of five N-linked 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. For site-specific glycan analysis, the fusion protein was hydrolyzed with trypsin, and the resulting tryptic digests were analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). The expected N-linked glycosylation sites were successfully identified and site-specific glycopeptide mapping was completed by Integrated GlycoProteome Analyzer (I-GPA) for the resulting raw tandem mass data. Finally, it was clearly confirmed that N-linked glycans for each glycosylation site showed significantly different patterns in microheterogeneity, which may indicate certain functions for each glycosylation site in the protein. Based on the mapping results, the unique features in glycan microheterogeneity for the five glycosylation sites of VEGFR-IgG fusion protein were compared site-specifically and further discussed to understand the functional meaning of each glycosylation pattern.

Highlights

Worldwide development of biosimilar drugs has grown over the past decade in an attempt to decrease the burden of healthcare costs produced from costly biopharmaceutical drugs
The biosimilar VEGFR-IgG fusion protein was developed by fusing the homodimer of VEGFR-1 and VEGFR-2 with the human IgG Fc region
Fusion proteins are typically constructed by fusing the Fc portion of human IgG1 with extracellular domains of native transmembrane receptor proteins and often retain several N-glycosylation sites, including a single site at Asn297 in the Fc region of IgG1

Summary

Introduction

Worldwide development of biosimilar drugs has grown over the past decade in an attempt to decrease the burden of healthcare costs produced from costly biopharmaceutical drugs. Biosimilars are defined as biological medicinal products that are highly similar to and have no clinically meaningful differences from existing reference products. Many blockbuster IgG monoclonal antibodies and related Fc fusion proteins are currently reaching patent expiry allowing for the rise in biosimilar development [1]. Human Fc-fusion proteins have become a popular platform for biotherapeutics. Fusion proteins are typically constructed by fusing the Fc portion of human IgG1 with the extracellular domains of native transmembrane proteins linked to a target molecule [2]. Fusion proteins retain the function of the soluble receptor or ligand and have increased clinical performance due to the addition of the Fc portion of human IgG1, which allows for glycan-mediated clearance, improved protein stability and solubility, and IgG recycling for an extended half-life [3]

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