Abstract
Exenatide 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 (-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 ha/odurans
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.