Tuning selectivity in cation-exchange chromatography applied for monoclonal antibody separations, part 2: Evaluation...

In this second part of the series, recently commercialized cation exchanger stationary phases were systematically investigated for their capabilities to separate therapeutic monoclonal antibodies. It was demonstrated that the different combinations of stationary and mobile phases result in diverse retention, selectivity and efficiency. Hence, the whole phase system (combination of stationary and mobile phase) should be considered when developing a method. In addition, retention behavior is mAb dependent and should be individually optimized. Another interesting observation was that in cation exchange chromatographic separations of large proteins, the particle size of the columns probably impacts retention rather than efficiency, due to the non-porous particle structure - and therefore the higher specific surface area of smaller particles -. Particle size influences the specific surface area and total porosity. Therefore, columns packed with larger particles showed lower retention (when the ion exchanger group was the same e.g. strong exchanger sulfonic group) while no link was observed between efficiency and particle size. The retention, efficiency and selectivity of the studied columns were quite different and strongly dependent on the elution mode (i.e. salt gradient, pH gradient or combined salt/pH gradient mode). The columns can be considered to be complementary, suggesting that it is useful to have more than one type of column on hand while developing new charge variant assays. Moreover, this work shows that it is especially attractive to make use of short, narrow bore ion exchange columns that offer the possibility to perform 4-6 min long separations of both intact and partially digested antibodies.