Doping reversed-phase media for improved peptide purification.
The purification of therapeutic peptides is most often performed using one or more reversed phase chromatography steps. This ensures high purities while keeping the costs of purification under control. In this paper, a doped reversed phase chromatographic material is tested and compared to traditional reversed phase materials. The doping consists of adding limited amounts of ion exchange ligands to the surface of the material to achieve orthogonal separation and increase the non-hydrophobic interactions with the surface. These ionic groups can either be attractive (opposite charge), or repulsive (same charge) to the peptide. The benefit of this new doped reversed phase material is shown through increases in selectivity in diluted conditions and yield and productivity in overloaded (i.e. industrial) conditions. It is the conjectured that all performance characteristics should increase using repulsive doping groups, whereas these characteristics should decrease when using attractive doping groups. This conjecture is shown to be true through several examples, including purifications of industrially relevant peptide crudes, in industrially relevant conditions. Moreover, the effect of ionic strength and organic modifier concentration was explored and shown to be in line with the expected behavior.
Institute for Chemical and Bioengineering, ETH Zurich, 8093 Zurich, Switzerland.
Rushd
R
Khalaf
Zeochem AG, 8707 Uetikon, Switzerland.
Nicola
N
Forrer
Zeochem AG, 8707 Uetikon, Switzerland.
Gianluca
G
Buffolino
Institute for Chemical and Bioengineering, ETH Zurich, 8093 Zurich, Switzerland.
David
D
Gétaz
Institute for Chemical and Bioengineering, ETH Zurich, 8093 Zurich, Switzerland.
Susanna
S
Bernardi
Institute for Chemical and Bioengineering, ETH Zurich, 8093 Zurich, Switzerland.
Alessandro
A
Butté
Institute for Chemical and Bioengineering, ETH Zurich, 8093 Zurich, Switzerland. Electronic address: massimo.morbidelli@chem.ethz.ch.
Massimo
M
Morbidelli
D056148:Chromatography, Reverse-Phase / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*
D008024:Ligands; D010455:Peptides
Doped reversed phase; Ion exchangers; Mixed mode; Overloaded conditions; Peptide purification; Reversed phase