Characterisation of reversed phase chromatography peptide separation systems

With the advancement of the biopharmaceutical industry, it is imperative to have a firm understanding of the peptide separation system. The chromatographic profile can be extremely complex for peptides with various impurities or degradation products, thus it is essential to be able to maximise selectivity differences to identify each species. This thesis endeavoured to understand the influencing factors to enable rational decisions to be made during method development strategies.;A peptide-based characterisation protocol was developed and applied to commercialised stationary phases, known as the Peptide RPC Column Characterisation Protocol. A design of experiment robustness study was executed to ensure the validity of the protocol was maintained. Chemometric analysis was performed which identified three classifications of columns from which stationary phases can be selected with either similar or different selectivity. The approach was validated using two tryptic digested peptides, with promising results.;Although there are small molecule characterisation protocols already defined in the literature, it was confirmed that the peptide-based protocol is needed due to a lack of correlation between the small molecule and peptide-based protocols.;A comprehensive mobile phase study on a typical C18 phase also identified vast selectivity differences over a range of pH values, which was achieved using different ammonium-based salts, with interesting additives and ion pairs. The study ascertained the greatest differences were achieved under low pH conditions, thus the main focus for method development should be in that region. An initial study which observed the effect of temperature and organic modifiers identified the importance of evaluating these parameters during method development. Finally, the applicability of the results on other columns was determined.;The conclusions can provide a firm platform to develop a comprehensive method development strategy, which should provide a more rational approach for screening relevant stationary phases and mobile phases to truly maximise selectivity differences.With the advancement of the biopharmaceutical industry, it is imperative to have a firm understanding of the peptide separation system. The chromatographic profile can be extremely complex for peptides with various impurities or degradation products, thus it is essential to be able to maximise selectivity differences to identify each species. This thesis endeavoured to understand the influencing factors to enable rational decisions to be made during method development strategies.;A peptide-based characterisation protocol was developed and applied to commercialised stationary phases, known as the Peptide RPC Column Characterisation Protocol. A design of experiment robustness study was executed to ensure the validity of the protocol was maintained. Chemometric analysis was performed which identified three classifications of columns from which stationary phases can be selected with either similar or different selectivity. The approach was validated using two tryptic digested peptides, with promising results.;Although there are small molecule characterisation protocols already defined in the literature, it was confirmed that the peptide-based protocol is needed due to a lack of correlation between the small molecule and peptide-based protocols.;A comprehensive mobile phase study on a typical C18 phase also identified vast selectivity differences over a range of pH values, which was achieved using different ammonium-based salts, with interesting additives and ion pairs. The study ascertained the greatest differences were achieved under low pH conditions, thus the main focus for method development should be in that region. An initial study which observed the effect of temperature and organic modifiers identified the importance of evaluating these parameters during method development. Finally, the applicability of the results on other columns was determined.;The conclusions can provide a firm platform to develop a comprehensive method development strategy, which should provide a more rational approach for screening relevant stationary phases and mobile phases to truly maximise selectivity differences