The development of a sub/supercritical fluid chromatography based purification method for peptides.

Abstract Peptide drugs are essential components of the pharmaceutical industry with a multiplicity of therapeutic properties, such as being anti-hypertensive, anti-microbial, anti-diabetic, and having anti-cancer potential. These molecules are similar in physiological structure and function to the body's endogenous signalling molecules and are therefore ideal candidates for the development of the next-generation of drugs. However, the purification of these peptides can be problematic due to poor solubility and stability, which often results in low peptide yields. Peptides are traditionally purified via RP-HPLC methods, which are tedious and employ harsh solvents that generate harmful waste to the environment. There is a growing need for more cost-effective and sustainable purification methods of these biologics. SFC can provide a greener peptide purification approach with more environmentally friendly mobile phases such as CO2 and methanol, which can easily be recycled with minimal environmental impact. Currently, there is limited knowledge regarding the SFC purification of peptides. Herein, this study investigated SFC methods to purify a tetrapeptide (LYLV), octapeptide (DRVYIHPF), and nonapeptide (LYLVCGERG) on commercially available columns at an analytical scale. The 2-ethyl pyridine column proved to be optimal based on its reproducibility, peak shapes, efficient separations, and retention factors with peptide recoveries ranging from 80 to 102%. The run times were reduced to 13 min, as opposed to the traditional RP-HPLC methods of 50 min, thus making this SFC method an efficient, greener, and more cost-effective approach for the purification of these peptides.