Development of a rapid UHPLC method for the in-process determination of coupling reagents used in peptide synthesis

The following thesis details the extensive development of a rapid liquid chromatography method for the in-process determination of peptide coupling reagents used in peptide synthesis. The determination of peptide coupling reagents, additives and associated by-products is important during peptide synthesis to ensure the concentration of these products are below the threshold of toxicological concern in the final peptide. A number of column technologies and mobile phases were evaluated for the separation of the analytes using an ultra-high performance liquid chromatography system and, a 15 minute method for the simultaneous determination of fourteen peptide coupling reagents, additives and by-products was established. This method was determined to be selective and capable of accurately quantitating the amount of TMU, HOBt, HCTU, HBTU, 6-ChloroHOBt, TBTU, Oxyma Pure, COMU, DIU, DIC, PyBOP, and TCTU in the presence of two peptides from Ipsen Manufacturing Ireland LTD. Analytical method validation was performed as per ICH guidelines for specificity, accuracy, linearity, precision, detection limit, quantitation limit and robustness. This rapid UHPLC method is directly transferable onto LC-MS and the intended application of this method is for evaluation of development peptides in Ipsen Manufacturing Ireland LTD.