Steenkamp, Lucia HSteenkamp, P2026-01-052026-01-0520251520-572X1082-6076https://doi.org/10.1080/10826076.2025.2502034http://hdl.handle.net/10204/14561Exenatide is used for the treatment of type 2 diabetes. Exenatide is normally produced using solid or liquid phase chemical synthesis which requires protecting side chain groups. The C-terminal is then amidated while the protecting groups are still in place. The novel production of exenatide, using a microorganism with the correct coding sequence, does not require protecting groups. The exenatide is then amidated using a PAM enzyme system. An UPLC-QTOF-MS method was developed to separate and analyze the non-amidated exenatide molecule from the final active amidated exenatide. The non-amidated exenatide was produced using an expression construct comprising a carrier protein open reading frame (ORF) Yarrowia lipolytica lipase or truncated Bacillus halodurans flagellin cloned in frame with the coding sequence for exenatide. The non-amidated and amidated exenatide differ by 1 Dalton in mass and typically co-elute during analysis. The method developed was able to separate the two compounds and could be used to measure the amidated exenatide produced during the bioconversion. The aim of the study was to investigate the feasibility of producing exenatide and monitoring the amidation during the bioconversion for possible scale-up and commercialization. The results showed complete amidation of the glycine-extended heterologous exenatide. The reproducibility of the analytical method was evaluated and found that the retention times and peaks areas of the detected exenatide were stable, making this analytical method suitable for reaction monitoring.FulltextenType 2 diabetesUPLC-QTOF-MSAmidationAccurate massArticleN/A