Vydac® Reversed Phase Prep & Process Chromatography Application Note

A Powerful But Often Overlooked Method for Protein Purification

Reversed-phase chromatography on Vydac® 300Å pore-size adsorbents, first introduced in 1981, is a very useful high-resolution method for protein separations. The power of reversed-phase for protein chromatography is demonstrated, for example, by the separation of species-specific insulins, many of which differ by a single amino acid residue, the separation of various apolipoproteins from human serum, the separation of reduction products which differ mainly in conformation from native insulin, and the separation of ribosomal proteins.

Subtle differences in conformation can permit protein separation by reversed-phase chromatography because retention depends on the “hydrophobic footprint” of a protein molecule – the minority of hydrophobic amino acid residues that are actually accessible at the surface of the folded protein. Since sample loading occurs at low organic concentration, this can reflect subtle differences in native structure.

A rule of thumb for developing preparative and process purifications is that a method that succeeds in  revealing an impurity will often be the best method for removing that impurity. The frequent use of reversed-phase for protein analysis would therefore argue for its utility in purifications. To be fair, chromatography on Vydac® reversed-phase adsorbents is already used in process purification of several FDA-approved bio-pharmaceuticals, and new reversed-phase purifications of protein products are reported regularly. However it also appears that reversed-phase methodology, while highly regarded as an analytical method and in spite of its prodigious resolving power, is often dismissed or overlooked as a candidate method for purification.

Why is this?
We believe it reflects some apprehensions about reversed-phase that actually have simple answers.