New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: Theory and applications

In the pharmaceutical field, there is considerable interest in the use of peptides and proteins for therapeutic purposes. There are various ways to characterize such complex samples, but during the last few years, a significant number of technological developments have been brought to the field of RPLC and RPLC–MS.Thus, the present review focuses first on the basics of RPLC for peptides and proteins, including the inherent problems, some possible solutions and some directions for developing a new RPLC method that is dedicated to biomolecules. Then the latest advances in RPLC, such as wide-pore core–shell particles, fully porous sub-2 μm particles, organic monoliths, porous layer open tubular columns and elevated temperature, are described and critically discussed in terms of both kinetic efficiency and selectivity. Numerous applications with real samples are presented that confirm the relevance of these different strategies. Finally, one of the key advantages of RPLC for peptides and proteins over other historical approaches is its inherent compatibility with MS using both MALDI and ESI sources.

► RPLC is a powerful strategy for peptide and protein separations.
► The new wide-pore UHPLC and core–shell particles provide some impressive results even with large proteins.
► Elevated mobile phase temperature is an additional parameter for improving separations of large biomolecules.
► Trends in RPLC method development of proteins and peptides are presented.
► The most important advantage of RPLC over IEX is its inherent compatibility with ESI-MS.