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Overview of Fc-fusion Protein Analysis

Peptides and proteins are interesting drug candidates due to their important role in many different disease pathologies. However, the clinical potential of these biomolecules is often hampered by their inherent short serum half-life. This problem brought forth a unique class of therapeutics, namely, Fc-fusion proteins, that combine the beneficial pharmacological properties of biological ligands with the additional properties of the fragment crystallizable (Fc) domain of an immunoglobulin G (IgG). Indeed, the fusion of the IgG-Fc domain to a ligand, active peptide, or extracellular domain (ECD) of a receptor greatly improves the clinical potential of active protein drugs for example by extending the plasma half-life as well as engaging immune-mediated effector functions that may also be silenced.

Fig.1 Concepts of Fc-fusion therapeutic protein and its applications. (Hirasawa, 2019)

There is a very high structural diversity within the molecules belonging to this class, and although the Fc part is common to all these products, the second part of the Fc-fusion protein (i.e., biological ligand) can be highly diverse in terms of size, charge, and hydrophobicity (i.e., it can be either extracellular domains of natural receptors, functionally active peptides, genetically engineered binding constructs acting as cytokine traps or even recombinant enzymes). This diversity explains why specific chromatographic, electrophoretic, and MS methods must be developed for Fc-fusion proteins. Besides this, the glycosylation profile of Fc-fusion proteins is highly complex involving the presence of multiple N- and O-glycans sites and numerous sialic acids, requiring highly innovative and complementary analytical methods.

Fig. 2 Effect of glycosylation on the charge isomers. (Duivelshof, 2021)

Methods of Fc-fusion Protein Analysis

The structural complexity and heterogeneity of Fc-fusion proteins require a set of analytical tools to be properly characterized.

• The analytical methods were used to confirm the primary structure of the Fc-fusion proteins, to evaluate the main PTMs, such as charge/size variants and glycan profile, and the main physicochemical properties of Fc-fusion proteins, such as identity, purity, and integrity.
• The identity of an Fc-fusion protein can be determined by a variety of analytical methods including ion-exchange chromatography (IEX), imaged capillary isoelectric focusing (icIEF), hydrophobic interaction chromatography (HIC), reversed-phase liquid chromatography (RPLC), and peptide mapping using liquid chromatography-mass spectrometry (LC-MS). Among them, IEX and icIEF are mostly used because the distribution of charge variants of an Fc-fusion protein provides a distinctive fingerprint of the protein.
• Purity and integrity confirmation of Fc-fusion proteins is required throughout all stages of manufacturing, storage, and administration to the patient. Like other therapeutic proteins, Fc-fusion proteins are susceptible to PTMs and degradation that could eventually result in fragmentation and aggregate formation. The purity assessment and size variant characterization are traditionally performed by size exclusion chromatography (SEC) or capillary electrophoresis sodium dodecyl sulfate (CE-SDS), by measuring the level of high molecular weight species, monomer, and fragments such as target peptide-Fc variants.
• The method used for the glycan analysis is usually through hydrophilic interaction chromatography (HILIC).

References

1. Hirasawa, S.; et al. Facile and Efficient Chemoenzymatic Semisynthesis of Fc-Fusion Compounds for Half-Life Extension of Pharmaceutical Components. Bioconjug Chem. 2019, 30(9): 2323-2331.
2. Duivelshof, B. L.; et al. Therapeutic Fc-fusion proteins: Current analytical strategies. J Sep Sci. 2021, 44(1): 35-62.