Preparative chromatography of peptides and other large molecules
STEVEN MCINTYRE
Peptide Process Development Department Manager, Almac, Seagoe Industrial Estate, Craigavon, United Kingdom
Abstract
Over the last number of years, analytical technologies for assessing the purity of a synthetic peptide have changed significantly, with U(H)PLC methods utilising media with specific properties designed to separate peptide impurities used in almost all peptide analyses. Furthermore, additional scrutiny of these methods via mass spectrometry or ion mobility spectrometry ensures that all impurities produced during the manufacture of a peptide are now routinely detected.
The use of preparative chromatography is still the main option for purification of peptides, a technology that has not quite advanced at the same rate, with 10µm C18 silica still the method of choice for most purifications. This paper will describe the holistic approach taken at Almac to ensure peptides and other large molecules produced for clinical and commercial purposes are still able to meet the stringent regulations and increased scrutiny.
REGULATORY REQUIREMENTS FOR SYNTHETIC PEPTIDES
Information in the European Pharmacopeia (EP) (1), as summarised in Table 1 highlights the required impurity control for synthetic peptides as they proceed through clinical development.
In general terms, control of impurities should increase through clinical development. Specifically, individual impurities should not exceed their values as qualified in toxicological studies, or be greater than 1.0%, whichever is higher. However, these limits apply to a specific analytical method and as method development is performed, this can result in changes to the levels of impurities previously thought to be qualified. Hence, it is important to perform analytical development with this in mind, and ensure knowledge of the overall impurity profile of a product is understood right from the start.
Analytical development and impurity detection
In analytical development, a screening approach is applied using material generated early in process development. Whilst no two peptide methods are alike, they typically encounter the same challenges:
