Timothy M. Pabst, University of Virginia, Charlottesville, VA 22904, Giorgio Carta, Department Chemical Engineering, University of Virginia, Charlottesville, VA 22904-4741, Alan K. Hunter, Global Biologics, Pfizer, 700 Chesterfield Parkway West, Chesterfield, MO 63017, and Natarajan Ramasubramanyan, Global Biologics, PGRD, Pfizer Inc, 700 Chesterfield Pkwy West, Chesterfield, MO 63005.
Complex pH transitions occur in exchange columns containing weak acid or weak base during salt steps or gradients if the mobile phase is buffered and the buffering species do not interact with the stationary phase. If not anticipated and planned for within a separation step, they can result in large equilibration volumes and potentially negative effects on pH-sensitive proteins. On the other hand, such pH variations, if controlled and directed, can be beneficial to the separation of proteins. The behavior of chromatographic columns packed with resins containing both weak cation exchange and weak anion exchange groups is investigated in order to obtain protein separations by means of internally generated pH gradients in response to step changes in buffer composition. A local equilibrium model is developed to predict pH transitions using non-adsorbed buffers, i.e. containing neutral or anionic buffering species with cation exchangers and neutral or cationic buffering species with anion exchangers. In agreement with experimental results, the model, based exclusively on the resins' titration curves, predicts practical, fairly linear gradients, which are formed using suitable mixtures of acidic buffers for cation exchange and basic buffers for anion exchange. The separation of mixtures of ovalbumin, albumin, and transferrin is used as a model system with cation exchange columns while the separations of transferrin and of mAb charge variants are used as model systems with anion exchange columns. The peaks obtained with this approach are much sharper than could be obtained isocratically or using externally generated, unretained gradients as a result of the peak compression caused by the axial pH gradient formed along the column. Moreover, separation is obtained at very low ionic strengths (2-3 mS/cm). The effects of flow velocity, mobile phase composition, time of injection, and protein load on retention and elution pH are investigated systematically demonstrating a range of ways in which the separation can be controlled and optimized.