Impact of mobile phase temperature on recovery and stability of monoclonal antibodies using recent reversed-phase stationary phases.

Recent reversed-phase wide-pore stationary phases were evaluated for the separation of intact monoclonal antibodies and their fragments. Two types of stationary phases were tested: Phenomenex Aeris Widepore, with 3.6 μm core-shell particles and Waters Acquity BEH300 with 1.7 μm fully porous particles. A systematic investigation was carried out using model IgG1 and IgG2 antibodies, namely rituximab, panitumumab, and bevacizumab. It appeared that adsorption of these antibodies on the stationary phase was significantly higher compared to proteins of equivalent size. The adsorption was particularly important for the intact antibodies of 150 kDa and for the largest fragments of 50 to 100 kDa (i.e., heavy chain, -fraction of antigene-binding). The present study demonstrated an obvious relationship between adsorption phenomenon and the unwanted strong secondary interactions (ionic and hydrogen bond) of the stationary phase. Thus, adsorption was more pronounced on the Aeris column because of the stronger ion exchange contribution of this stationary phase. Using C4 phase instead of C18 at 50-70°C, there is a slight reduction (5-20%) in adsorption. Two solutions were proposed to decrease the strength of secondary interactions and thus resolve (or at least diminish) adsorption issue. First, increasing mobile phase temperature up to 80-90°C appeared as a promising solution. However, temperature should be used with caution as it can partially damage large biomolecules. A compromise between residence time and temperature should be found. Second, it is recommended to add a small amount of an ancillary solvent, such as n-butanol to the mobile phase. Indeed, the hydroxyl group of n-butanol probably interacts with water adsorbed on the residual silanol groups "to shield" silanols.