This work explores the potential of supercritical fluid chromatography for the analysis of peptide drugs. The regulatory aspects of peptides are more complex and challenging than for small drug molecules. The same applies to the analytical characterization of peptides. After technical innovations and the introduction of a new instrument generation, supercritical fluid chromatography nowadays is a suitable alternative to reversed-phase chromatography, which is considered the gold standard in peptide analysis. However, the scientific data on the analysis of peptides with supercritical fluid chromatography is limited. In the first part of this thesis, supercritical fluid chromatography is used to develop a method to separate the variety of cyclic and linear peptides characteristic for tyrothricin. A software-based design of experiments approach is utilized to optimize an analytical method that provides superior separation of cyclic peptides compared to the reversed-phase separation. Even isomeric peptide pairs are efficiently separated. Subsequently, the potential of a binary mixture of methanol and acetonitrile combined with additives as the modifier for the separations of other cyclic peptides is investigated. The proportion of the mixture is optimized to improve the resolution and control the elution order of the analytes. Paired with an aromatic stationary phase, this chromatographic parameter offers an effective method optimization opportunity. This knowledge is furthermore extended to larger and more hydrophilic peptides in the following experiment. A single method for the purity analysis of human insulin and six analogs is optimized. A crown ether is also investigated as a promising additive to control the elution order of the insulins in dependence to its concentration. In the last study, the applicability of an established chromatographic modeling software for the in-silico optimization of peptide separations in supercritical fluid chromatography is demonstrated. Optimizing a binary mixture of methanol and acetonitrile serves as an effective parameter of the model. Using such software tools offers enormous time savings and, at the same time, meets the current requirements for analytical method development according to the quality by design concept. These already published studies are finally put into the context of the life cycle concept of analytical methods that will soon be demanded by the ICH Q14 guideline "Analytical procedure development". Existing knowledge, in the form of scientific publications, is employed as the basis for a general risk assessment applicable to peptide separations with supercritical fluid chromatography. From this, a generic method development strategy is derived and presented. This way, the current state of science and technology is taken into account to define an efficient workflow. This is particularly relevant in early pharmaceutical development phases, from where only a small fraction of initial drug candidates reaches market maturity. At the same time, this approach enhances the drug knowledge in early development phases and ultimately serves patient safety. The present work makes an important contribution to demonstrating the potential of supercritical fluid chromatography for the analysis of peptide drugs. It serves as a concise example of how supercritical fluid chromatography can be interpreted as a new technique in the ICH Q14 guideline setting. The approach presented can be easily applied to any other analytical technique or analyte class.
