Session: Session 2

Questioning chirality in therapeutic peptides: Effect histidine residue ?

Controlling chirality within therapeutic peptides is major issue [1] as there is no guarantee that the stereogenic centre of an amino acid (naturally in L form) will not be altered during synthesis,storage or post-administration. This work aims to develop LC/EC-MS coupling methods for detecting and quantifying the optical alteration of His residue in therapeutic peptides, with an accuracy of 0.1%, in line with international regulations for the determination of impurities in the final product.

Thus, sets of model tripeptides containing a His residue (in L and D forms) with varying sequential positioning were synthesised. Histidine was selected due to its susceptibility to this optical conversion, based on the reactivity of its side chain [2].

Using LC-UV and CE-UV methods we were able to differentiate peptides containing a L-His residue from its counterpart containing a D-His residue. Using LC-UV in isocratic mode, we also managed to differentiate the model tripeptides based on the position of the His residue within the peptide sequence. LC-UV and CE-UV, do not provide the sensitivity required by international regulations, so coupling these technologies with MS is essential. With this in mind, preliminary studies in MS/MS were carried out.Peptides were cationized by alkali metals, used as chelating agents, then subjected to collision induced dissociations (CID) in an attempt to differentiate peptides containing an L-His residue from their counterparts containing a D-His residue.[3] Provided that stereospecific fragmentation routes will be achieved, Energy resolved mass spectrometry (ERMS) experiments and the survival yield technique will be performed in an effort to separate epimeric peptides. The development of an LC/CE-MS method using these strategies would become an incredibly powerful tool for the pharmaceutical industry.

 

[1] Ceramella, J. et al, Appl. Sci. 2022

[2] W. Van Den Nest et al., J Pept Sci,7, 2001

[3] Memboeuf et al, J. Am. Soc. Mass Spectrom. 22 (2011)