Characterization of low-level D-amino acid isomeric impurities of Semaglutide using liquid chromatography-high resolution tandem mass spectrometry.

Under the guideline issued by Food and Drug Administration (FDA), ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin Guidance for Industry, a synthetic Semaglutide that is intended to be a "generic" of the approved rDNA origin Semaglutide is under exploring. Thus, each peptide-related impurity that is 0.10% of the drug substance or greater need to be identified for Semaglutide covered by this guidance. Among others, characterization of the low-level D-amino acid (D form) isomeric impurities are always the most challenging ones. Reverse-phase high-performance liquid chromatography (RP-UPLC) was used to separate the impurities, followed by high resolution mass spectrometry (HRMS) to determine the molecular weight of the impurities that existed in both formulations. Following the targeted D form isomers off-line collection, the samples went through lyophilization, deuterated hydrochloric acid (D-HCl) hydrolyzation with low level D/L form shifting suppression substrates, chiral derivatization and RP-UPLC tandem mass spectrometry analysis of different amino acids by comparing with standards. Herein, we reported an accurate, straightforward characterization method with low limit of detection for the low-level D-Ser, D-His and D-Asp Semaglutide impurities in Semaglutide formulations. The developed UPLC tandem HRMS method entails a valuable step forward in the detection of trace levels of the D-isomers of Semaglutide and other peptide products.