Understanding Separation Parameters For Intact Protein LC/MS Analysis Using Wide-Pore Core-Shell Media — ASN Events

Understanding Separation Parameters For Intact Protein LC/MS Analysis Using Wide-Pore Core-Shell Media (#268)

Hung Luu 1 , Duncan Macinnis 1 , Aman Sharma 1 , David Shock 1
  1. Phenomenex Australia, Lane Cove, NSW, Australia

Previous reports have shown that diffusion rates of proteins are inversely proportional to the log of the molecular weight of a protein; data confirms that slow diffusion of large proteins in and out of a porous particle is the limiting factor in minimizing peak width of a protein in reversed phase chromatography. The newest generation of core-shell media utilizes thin porous layers to minimizing band dispersion while maintaining strong shape selectivity; separation of closely related modified proteins demonstrate similar separation with a concomitant reduction in protein peak width based on the shell thickness of the core-shell particle. Optimizing shell thickness is a balance between loading and performance; a thinner shell has a shorter diffusion path with lower surface area, loading, and retention. A thicker shell allows for greater loading and retention but reduces performance.

Results show that the lower retentivity of the thin shell product requires significant reduction in organic content during gradient methods (resulting in subsequent reduction in electrospray efficiency); however, the increased recovery and efficiency of wide-pore core-shell columns far outweighs such losses with an approximate 50 % increase in MS sensitivity for evaluated protein samples. Optimized methods for IgG antibodies demonstrate separation of major disulfide-related isoforms with significant increases in protein recovery. Resolution of disulfide isoforms studies suggest that such applications retain ability to partition proteins based on some degree of shape selectivity which is especially useful in therapeutic protein characterization. The reduced retention of these media allow the use of acetonitrile as the organic mobile phase (versus IPA mixtures for fully porous media) as well as lower column temperatures which allow for different selectivity and resolution of post-translational modifications.