Fc-fusion Purification Performance: Amsphere™ A3 Versus Other Protein A Resins

Fc-fusion proteins are immunoglobin Fc domains linked to proteins with therapeutic potential. These can favorably increase serum half-life and alter a biopharmaceutical’s immunological properties. There are currently 13 commercially approved Fc-fusion biopharmaceuticals and many more in clinical trials (1, 2). Capture chromatography of Fc-fusion proteins on Protein-A based affinity chromatography resins is a favored method of purification.

However, Fc-fusion proteins often exhibit lower dynamic binding capacities (DBCs) than regular monoclonal antibodies (mAbs) (3, 4). Two factors that may contribute to this are decreased Fc-domain affinity (5) or that some Fc-fusion proteins are larger than mAbs (4). Given the wide range of commercial Protein A-based affinity resin matrices (e. g., agarose and polymethacrylate), and their properties such as average particle and pore sizes, it is not surprising to see significant affinity chromatography performance differences (4). This suggests that bioprocess scientists should screen various resins with their targets. Amsphere A3 (A3) is a Protein A resin offering relatively small average particle size (50 μm), large average pore size (46 nm)(4) and good target mass transport properties, including vertical breakthrough  performance (4), and sound large column pressure-flow properties. Its unique hydrophilized polymethacrylate particle chemistry allows A3 to typically exhibit high DBCs and good alkaline stability. It can be used to bind and purify a variety of antibody and Ab-derived molecules (6).

In this note, the DBC and purification of several Fc-fusion and bispecific Ab proteins are compared on A3 and other leading commercial Protein A resins. A3 generally showed the highest DBC values and comparable purification performance. The molecular basis for A3’s high affinity for these target molecules is discussed.