Identification and determination of structurally related peptide impurities in thymalfasin by liquid chromatography-high-resolution mass spectrometry.

Thymalfasin is an important peptide drug widely used for the single or combination treatment of hepatitis, sepsis, cancer, and immunodeficiency. Accurate purity assessment of thymalfasin material is essential for thymalfasin certified reference materials (CRMs) production and analytical method validation, in which comprehensive determination of thymalfasin-related impurities is required to avoid quantitative bias. In this study, liquid chromatography-high-resolution mass spectrometry (LC-hrMS) methods have been established to comprehensively characterize and quantify thymalfasin-related impurities using a thymalfasin China Pharmacopoeia (ChP) standard and then successfully applied to three commercial thymalfasin materials. A total of twenty-three thymalfasin-related impurities (> 0.1 mg/g) were separated, identified, and quantified in the ChP standard analyzed. The major impurities existing in thymalfasin ChP standard and commercial materials include deamination, amination, succinimide, amino acid insertion/deletion, dimers, and isomers at different mass fraction levels. In particular, over half of the thymalfasin-related impurities were found directly or indirectly arising from the labile C-terminal asparagine (Asn) residue. Given the 28th Asn residue at the C-terminus is not necessary for the biological activity of thymalfasin as reported previously, thus deletion, replacement, or modification of thymalfasin C-terminal Asn residue is proposed for new drug research and development. In summary, these results provide a further complement to the thymalfasin-related impurity profile and issue a warning for protection or processing of the thymalfasin C-terminal Asn residue.