Key considerations are described for the development and optimisation of an HPLC-based analytical method for evaluating the serum stability of a CGRP antagonist peptide for migraine treatment. The two experimental designs used, Plackett–Burman design and Taguchi design, afforded a method with good resolution between the parent peptide and its major metabolite, allowing the determination of its half-life in human serum.
Evaluation of the stability of peptide drug candidates in biological fluids, such as blood serum, is of high importance during the lead optimisation phase. Here, we describe the optimisation and validation of a method for the evaluation of the stability of a lead calcitonin gene-related peptide antagonist peptide (P006) in blood serum. After initially determining appropriate peptide and human serum concentrations and selection of the quenching reagent, the HPLC method optimisation used two experimental designs, Plackett–Burman design and Taguchi design. The analytical method was validated as complying with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. The optimised method allowed the successful resolution of the parent peptide from its metabolites using RP-HPLC and identification of the major metabolites of P006 by mass spectrometry. This paradigm may be widely adopted as a robust early-stage platform for screening peptide stability to rule out candidates with low in vitro stability, which would likely translate into poor in vivo pharmacokinetics.
