Peptides and proteins are interesting drug candidates due to their important roles in many different disease pathologies. However, the clinical potential of these biomolecules is often hampered by their short serum half-lives.
Fc-fusion protein analysis’ structural variability and complexity require the use of a variety of analytical techniques.
The basic physicochemical characteristics of Fc-fusion proteins, such as identity, purity, and integrity, as well as the main PTMs, including charge/size variations and glycan profiles, were assessed using the analytical methodologies.Several analytical techniques, such as 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), can be used to identify an Fc-fusion protein. Because an Fc-fusion protein’s distribution of charge variations acts as the protein’s unique fingerprint, IEX and icIEF are the two that are most frequently employed among them.
Fc-fusion proteins must be verified for purity and integrity at all phases of production, storage, and patient administration. Fc-fusion proteins are sensitive to PTMs and degradation, which could ultimately lead to fragmentation and aggregation formation, just like other therapeutic proteins.
By assessing the amount of high molecular weight species, monomers, and fragments such as target peptide-Fc variations, size exclusion chromatography (SEC) or capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is usually used to assess purity and characterize size variants.
Hydrophilic interaction chromatography (HILIC) is often the technique utilized for glycan analysis.