Two-dimensional Capillary-based Electrophoretic Separation Coupled to Mass Spectrometry by a Mechanical Valve

Abstract

Separation and identification are core disciplines in bioanalytics e.g. protein characterization. Capillary electrophoresis (CE) is a valuable technique for the analysis of intact proteins especially for the separation of their variants. Mass spectrometry (MS), providing high mass resolution and possible fragmentation experiments enables detailed identification and characterization of an analyte. However, low injection volumes and MS interfering components (ampholytes by applying capillary isoelectric focusing (CIEF) and involatile buffer utilizing capillary zone electrophoresis (CZE)) are in many cases drawbacks of CE separations. Tackling this drawbacks can be done in multidimensional setups. However, hyphenation of CE with CZE or HPLC is challenging due to high voltage, low transfer volumes and the need of dead volume free connections. The general objective of this work was to enable an interference free MS detection for electromigrative separation systems, with focus on CIEF as a first dimension, which are interfering the electrospray ionization (ESI) process when hyphenated directly to ESI-MS. A thorough literature research on CIEF online hyphenated to MS and their applications was performed and revealed the demands, challenges but also the power of such a coupling. This literature research was the foundation for the development of the here presented multidimensional setups and placed the focus on the main objective of this work: the CIEFCZE- MS hyphenation. For the practical part, a 4-port-valve was utilized, which allows the coupling of CE and CZE-MS. Starting with model samples containing proteins and peptides, important parameters of the CE-CZE-MS setup were evaluated, e.g. a procedure to find the best transfer volume (between 4-20 nL) or the introduction of a multiple heartcut approach (up to 6 cuts in one run). Consequently, different applications like the separation and identification of charge variants of a monoclonal antibody by CIEF-CZE-MS and CZE-CZE-MS, utilizing the aforementioned 4-port-valve followed the establishment of the system. It was possible to characterize deamidation variants of a monoclonal antibody on intact level in a fast and straight forward way. After the detailed evaluation of the aforementioned valve, it was also utilized for the promising nano HPLC(UV)-CZE-MS hyphenation for the separation of proteins and their corresponding variants. This combination of chromatographic and electromigrative separation mechanism was applied for the separation of a model protein mix in the first dimension. One of the obtained signals was detected as RNaseA/B in the LC(UV) dimension. This signal was further transferred into the CZE-MS dimension which enables the separation and MS detection of RNase A and RNase B including the different high-mannose variants of RNase B. The main goal of this project was the development of different multidimensional setups including electromigrative separation for the application e.g. in the bio pharmaceutical field. This was successful especially in the case of the charge variant characterization of a monoclonal antibody on intact level. Moreover the intensive work on the coupling device allows further applications like with the mentioned nano HPLC-CZE-MS setup which will be pursued in following projects. VII