Characterization of Glycosylation in Fc-Fusion Protein Drugs

Characterization of Glycosylation in Fc-Fusion Protein Drugs

CD BioGlyco has been committed to glycosylation research for many years, and we provide high-quality intact protein and subunit level analysis, intact glycopeptides analysis, glycan analysis, and sialic acid analysis services for our clients worldwide.

Fc-fusion Protein

The first Fc-fusion protein was reported in 1989, which is a new type of protein produced by fusing the Fc segment of immunoglobulin with certain biologically active functional protein molecules such as receptors, active peptides, and cytokines. Fc-fusion proteins not only exert the biological activity of fusion proteins, but also cause antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). In the pharmaceutical industry, Fc-fusion proteins have been used in drugs and vaccines. Protein drugs have a short half-life in plasma. To achieve therapeutic effects, frequent dosing is required to maintain clinically effective drug concentrations. The emergence of Fc-fusion proteins just solves this problem, which combines the beneficial pharmacological properties of biological ligands with the properties of the Fc domain of immunoglobulins, greatly improving the clinical potential of active protein drugs.

The structure of Fc-fusion protein.Fig.1 The structure of Fc-fusion protein. (Czajkowsky, et al., 2012)

Glycosylation of Fc-fusion Protein

Glycosylation is an important post-translational modification that plays an important role in the immunogenicity and clinical efficacy of therapeutic proteins. Unlike monoclonal antibodies, Fc-fusion proteins typically have multiple N-glycosylation sites and additional O-linking glycosylation sites in the biological domain in addition to containing a typical N-linked glycosylation site in the CH2 domain. These oligosaccharides have more complex bi-, tri-, and tetra-antenna structures. This complex glycosylation affects the in vivo clearance of Fc-fusion proteins via sialic acid content and terminal N-acetylglucosamine residues.

Our Services

Comprehensive glycosylation analysis of Fc-fusion proteins is critical due to its important effect on in vivo clearance. At CD BioGlyco, our characterization services of Fc-fusion proteins are the following:

  • Intact protein and subunit level analysis

We use appropriate enzymes to digest the intact proteins into relevant fragments and analyze them by electrospray ionization-mass spectrometry (ESI-MS) to obtain protein molecular weight information and data on major glycoforms.

  • Analysis of intact glycopeptides

We digest glycoproteins using different proteases to yield peptides and glycopeptides of approximately 0.5-5 kDa. The obtained peptides and glycopeptides are then analyzed by matrix-assisted laser desorption/ionization-MS (MALDI-MS) or ESI-MS. Finally, the peptide sequence and N-glycan linkage site are determined by the resulting peptide fragment ions and mass shifts generated by the attached glycans.

  • Glycan analysis

We use peptide-N-glycosidase F (PNGase F) to enzymatically remove N-glycans from glycoproteins, which are then fluorescently labeled with 2-aminobenzamide (2-AB) and analyzed by hydrophilic interaction liquid chromatography (HILIC).

  • Sialic acid analysis

Anion-exchange reversed-phase liquid chromatography (AEX-RPLC) method has been reported to analyze mono-sialylated, di-sialylated, and tri-sialylated polysaccharides.

CD BioGlyco provides high-quality, cost-effective, and hassle-free glycosylation characterization services in Fc-fusion protein drugs. If you are interested in our services, please feel free to contact us for more information.

References:

  1. Duivelshof, B.L.; et al. Therapeutic Fc-fusion proteins: Current analytical strategies. Journal of Separation Science. 2021, 44(1): 35-62.
  2. Czajkowsky, D.M.; et al. Fc‐fusion proteins: new developments and future perspectives. EMBO molecular medicine. 2012, 4(10): 1015-1028.
This service is for Research Use Only, not intended for any clinical use.

Christmas 2024

About Us

CD BioGlyco is a world-class biotechnology company with offices in many countries. Our products and services provide a viable option to what is otherwise available.

Contact Us

Copyright © CD BioGlyco. All rights reserved.
0