Temperature dependence of antibody adsorption in protein A affinity chromatography.

Staphylococcal protein A affinity chromatography is a well-established platform for purification of clinical-grade antibodies. The wild type ligand has been mutated to improve caustic stability, elution behavior, and/or to increase binding capacity. Several modified protein A ligands are nowadays commercially available, one of them being the thermosensitive chromatography medium Byzen Pro from Nomadic Bioscience Co., Ltd. According to the manufacturer, Byzen Pro has the ability to release IgG upon a change in temperature. It is based on a thermosensitive mutant of protein A which should allow elution at neutral pH by changing the temperature from binding at 5 °C to elution conditions at 40 °C. We determined equilibrium binding capacities of the thermosensitive protein A medium (Byzen Pro), MabSelect SuRe (GE Healthcare), and TOYOPEARL AF-rProtein A HC-650F (Tosoh Bioscience LLC) for antibodies of the subclass IgG1 and IgG2 at five different temperatures from 4 °C to 40 °C to elucidate the temperature effect. We also observed a temperature dependence of the dynamic binding capacities which were determined for the subclass IgG2 at three temperatures from 4 °C to 40 °C. However, for Byzen Pro, the temperature dependence was only present at a low flow rate and vanished at high flow rates indicating that pore diffusion is the rate-limiting step. Binding of the antibody to MabSelect SuRe and TOYOPEARL AF-rProtein A HC-650F stabilized the conformations as shown by an increase in melting temperature in differential scanning calorimetry measurements. The antibody conformation was slightly destabilized upon binding to the thermosensitive ligand. The conformation change upon binding was fully reversible as shown by circular dichroism, differential scanning calorimetry and size exclusion chromatography. Isothermal titration calorimetry was used to measure the raw heat of adsorption for the IgG2 molecule. The thermosensitive ligand can also be used for antibodies with low stability, because elution can also be effected by salt.