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Aggregation is among the most critical parameters affecting the pharmacological and safety profile of peptide Active Pharmaceutical Ingredients (APIs). For this reason, it is of utmost importance to define the exact aggregation state of peptide drugs, particularly when the API is marketed as a ready-to-use solution. Consequently, appropriate non-destructive techniques able to replicate the peptide environment must be employed. In our work, we exploited Asymmetrical Flow Field-Flow Fractionation (AF4), connected to UV, dRI, fluorescence, and MALS detectors, to fully characterize the aggregation state of Liraglutide, a peptide API used for the treatment of diabetes type 2 and chronic obesity. In previous studies, Liraglutide was hypothesized to assemble into hexa-octamers in phosphate buffer, but no information on its behavior in the formulation medium was provided up to now. The method used allowed researchers to work using formulation as the mobile phase with excellent recoveries and LoQ/LoD, discerning between stable and degraded samples, and detecting, when present, aggregates up to 108 Da. The native state of Liraglutide was assessed and found to be an association into pentamers, with a non-spherical conformation. Combined to benchmark analyses, the sameness study was complete and descriptive, also giving insight on the aggregation process and covalent/non-covalent aggregate types. [ABSTRACT FROM AUTHOR]

To understand the role of native aggregation state (NAS) of soluble wheat gluten and fractions during deamidation in a carboxylic acid/heat water solution, changes in conformation and deamidation behavior as function of protein concentration from dilute to semi-concentrated regimes to control NAS were investigated by physicochemical properties, SDS-PAGE, molecular force change, intrinsic fluorescence emission spectroscopy (IFES) and FTIR. Our data show that, in this solution, the deamidated proteins displayed features characteristic of more scattered and flexible polymer structure in dilute concentration than concentrated ones. Degree of deamidation (DD), HD and Zeta potential exhibited strongly oppositely with the decreasing concentration. HWM-GS, ω-gliadins and LWM-GS degraded into smaller peptides with decreasing of NAS. FTIR and IFES displayed that improved molecular flexibility with decreasing of concentration as detected by the increasing content of β-turn and β-sheet, as well as the red-shift of wheat gluten and gliadins at the expense of α-helix. Hydrophobic and hydrogen bond increased gradually and were dominant in inter-molecule as function of increasing concentration. The above information demonstrated that NAS of soluble wheat gluten dominated changes of deamidation behavior and conformation in a carboxylic acid/heat water solution. [ABSTRACT FROM AUTHOR]