Development of Reverse Phase Ultra-High Performance Liquid Chromatography for the Identification of Semaglutide

Abstract

The biopharmaceutical industry seeks to look for advanced analytical techniques to improve the quality of medicinal products. RP-UHPLC, or reverse phase ultra-high performance liquid chromatography, is a powerful tool for the precise detection of biological compounds.

The objective of the study was to develop a precise and sensitive RP-UHPLC method for peptide mapping of semaglutide that resembles a glucagon-like peptide-1 receptor agonist used to treat type 2 diabetes mellitus. The method development phase involved the optimisation of fundamental parameters, including incubation time, column temperature, and enzyme volume to achieve reliable and precise peptide separation. The chromatographic setup comprised of Waters XBridge C18 ODS column. The RP-UHPLC method was established using a 1 mg/mL stock solution of Semaglutide. The results revealed that the optimal conditions established were 6 hours of incubation time with column temperature at 37°C and 20μL of Glu-C endopeptidase. Peptide mapping results demonstrate remarkable results with high accuracy of peak separation and retention time for six specific Semaglutide peptides. This robust analytical approach suggests valuable insights for the characterization of therapeutics in pharmaceutical formulations. Moreover, the potential applicability of this method for analyzing other therapeutic proteins reflects its importance in advancing the pharmaceutical research and development sector.