Optimized superficially porous particles for protein separations

Continuing interest in larger therapeutic molecules by pharmaceutical and biotech companies provides the need for improved tools for examining these molecules both during the discovery phase and later during quality control. To meet this need, larger pore superficially porous particles with appropriate surface properties (Fused-Core(®) particles) have been developed with a pore size of 400 Å, allowing large molecules (<500 kDa) unrestricted access to the bonded phase. In addition, a particle size (3.4 μm) is employed that allows high-efficiency, low-pressure separations suitable for potentially pressure-sensitive proteins. A study of the shell thickness of the new fused-core particles suggests a compromise between a short diffusion path and high efficiency versus adequate retention and mass load tolerance. In addition, superior performance for the reversed-phase separation of proteins requires that specific design properties for the bonded-phase should be incorporated. As a result, columns of the new particles with unique bonded phases show excellent stability and high compatibility with mass spectrometry-suitable mobile phases. This report includes fast separations of intact protein mixtures, as well as examples of very high-resolution separations of larger monoclonal antibody materials and associated variants. Investigations of protein recovery, sample loading and dynamic range for analysis are shown. The advantages of these new 400 Å fused-core particles, specifically designed for protein analysis, over traditional particles for protein separations are demonstrated.