Moving beyond preparative reverse phase HPLC for peptide purification
Today’s peptide chemist has at his or her disposal a wide variety of chemistries to help make peptide synthesis more effective. There is a seemingly endless choice of coupling agents to help difficult couplings, or orthogonally reactive protecting groups to enable peptide side chain modification. But the methodology for purification has not been developed so readily – by far, the greatest reliance remains on preparative reverse phase HPLC (1). This is not because other techniques do not exist, but they have not been so readily adopted. This article examines the options beyond HPLC when it comes to peptide purification.
The interest in peptides as therapeutics shows no sign of slowing (2), and as a result, there is increased pressure on manufacturing capacity. The picture is complicated further by the fact that peptide therapeutics are taking on ever-more complex designs creating increased manufacturing challenges.
Short peptides, less than 5 amino acids or so, that are more akin to the molecular weight of small molecule therapeutics, stand a chance of being purified by non-chromatographic techniques such as precipitation or crystallisation. The methodology used for the purification of larger synthetic peptides is dominated by preparative reverse phase high performance liquid chromatography (RP-HPLC). RP-HPLC has proven to be a robust method of purification that is suitable for many circumstances, but it is not a universal technique that is guaranteed to be suitable for all occasions, and is often best used in partnership with other techniques.
This article looks at the advantages and disadvantages of RP-HPLC, and suggests some alternative methods that can be considered.