Characterization of protein based biotherapeutic drugs by ion mobility mass spectrometry

In recent years, the importance of protein-based therapeutic drugs has increased significantly. The fact that these drugs are produced from cell culture media or certain living things distinguishes these drugs from chemically synthesized drugs. These are drugs with therapeutic properties used to treat diseases such as cancer and diabetes. The structure must be unique for these drugs to play an effective therapeutic role against diseases. These drugs go through many critical stages during production and approval. Since such drugs are protein-based, the enzymatic and chemical modifications that occur during their production and post-production stages can vary in the functions and structures of these drugs. Such modifications that arise in their structures following protein synthesis are called post-translational modifications. Among these modifications, glycosylation is one of the most important ones. Mass spectrometry is a powerful analytical method used to elucidate many properties of protein-based therapeutic drugs such as sequence, composition, structure, conformation, and mass. Mass spectrometry is intensely used in the analysis of protein-based biotherapeutic drugs. However, these analytical techniques cannot discriminate species in analysis, especially in forms with the same mass/charge ratio and different conformational features. Ion mobility-mass spectrometry with soft ionization techniques plays an essential role in analyzing species with different conformational properties, especially with the same mass/charge ratio. Due to the soft ionization technique, ions can enter the mobility tunnel separated according to their shape, size, and charge characteristics. Thus, information about their conformational properties can be obtained. In this thesis, conformational N-glycan profile analyses of immunoglobulin G (IgG) in human serum, trastuzumab, and bevacizumab monoclonal antibodies (mAbs) were performed. In these analyses, the trapped ion mobility spectrometry (TIMS) technique was used as one of the ion mobility techniques highly compatible with mass spectrometers. The procainamide label was chosen for glycan analysis due to its advantage in ionization in mass spectrometric devices. In these analyzes, hydrophilic interaction chromatography was carried out with fluorescence and ion mobility-mass spectrometry detection techniques simultaneously. In the analyzes, glycan forms of mAbs were obtained and their conformational glycan structures were determined. The collision cross-section (CCS) values of each glycan form in the structures were calculated and compared. Especially, it was determined that the variation of CCS values of the glycan forms mainly arises due to the presence and binding formations of galactose and fucose units in their structures. In this thesis, information about the conformation of the glycan forms of mAbs was obtained using a TIMS device and a fluorescence detector simultaneously for the first time. A robust analytical method has been introduced to the literature.