ABSTRACT
Reversed-phase liquid chromatography (RPLC) is an essential tool for analytical and preparative separations of biopharmaceuticals. Its ubiquitous application in the discovery and development of protein-based drugs arises from its high resolving power, the robustness of the separation technique, and its success in handling a wide range of separation problems. The favorable kinetics provided by hydrocarbonaceous ligands bonded to microparticulate packings typically generate column efficiencies in excess of 25,000 plates/m. Silica-and polymer-based supports are stable over a wide range of solvent chemistries and operating pressures, and the hydrophobic stationary phases are quite robust compared to other chromatographic ligands. Reversed-phase columns are available in a wide range of dimensions for applications ranging from nanoscale analytical separations to process-scale chromatography, allowing RPLC methods to be scaled during product development. The technique usually employs volatile solvents, enabling easy solvent removal in preparative applications and allowing direct coupling to mass spectrometric detectors for analytical LC-MS. Most important, a vast array of mobile-phase chemistries and commercially available RPLC columns can be brought to bear on a separation problem. However, in practice, the success of a few generic methods for proteins and peptides usually simplifies the methoddevelopment task.
