There has recently been a renewed interest in peptide-based drugs due in part to advancements in overcoming short half-lives and the potential for new modalities. Therapeutic peptides can be made through both recombinant and synthetic strategies, but synthetic peptides can offer manufacturers the advantage of a regulatory approval process that follows that of a small molecule. When following a synthetic approach, process-related impurities can arise from the raw materials as well as the manufacturing process, and thus can differ from batch to batch or between manufacturing sites. LC-UV-based methods are most commonly used to determine product purity, but by incorporating a single quadrupole mass detector into the analysis, method development and optimization can be streamlined.
In this study, eledoisin, a vasodilator, is used as a clinically relevant sample to demonstrate how a single quadrupole mass detector can be used to streamline the method development process for synthetic peptide impurity monitoring. A general gradient method was used to screen multiple column chemistries in mobile phase containing 0.1% (v/v) trifluoroacetic acid or 0.1% (v/v) formic acid. This allows for critical attributes to be readily monitored, but can also help determine an initial set of chromatographic conditions. After selecting a column chemistry and mobile phase, a focused gradient can be used to separate additional impurities. Once a method is optimized, the added mass detection can identify co-elutions, which again is useful when a particular impurity compromises patient safety. This work demonstrates that by incorporating UPLC and orthogonal mass detection into a single workflow, there is an added level of assurance in product quality.