Capillary Zone Electrophoresis, Capillary Isoelectric Focusing, and Ion Exchange Chromatography for the Separation of Antibody Variants on the Intact and Subunit Level with Online Mass Spectrometric Characterization

Abstract

The analysis of monoclonal antibodies (mAbs) is an ever-evolving field of research. Depending on the host cell, the purification process, or different storage conditions, mAbs can differ, for example in their glycosylation profile or have alterations of amino acids. The analysis of these modifications is therefore quite complex and time-consuming and can generally be performed on an intact, subunit, or peptide level by coupling a separation technique with mass spectrometry (MS). Separation is key to detailed and unambiguous MS analysis of mAb variants, and various separation principles are used in the literature for this purpose. The general object of this thesis was to investigate capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and ion exchange chromatography (IEX) coupled with MS for their performance in analyzing charge variants of antibodies at the intact and subunit level. The newly developed CZE-MS method at the subunit level enabled the selective separation of several size and charge variants as well as multiple other minor variants (e.g. additional glycation) and reduced variants. The location of variants and positional isomers was determined by middle-down electron transfer higher energy collisional dissociation (EThcD). For the ten mAbs, fragmentation coverages between 10% and 30% were obtained and 10 – 40 mAb variants could be identified. Subunit moieties were additionally analyzed by ion mobility (IM) - MS and different ion mobilities for the different variants were obtained. For the first time, the orthogonality of CZE and IM was evaluated by analyzing a complex peptide mixture, and a very high orthogonality of around 80% was obtained. Additionally, to the successful CZE-MS subunit approach, mAbs were analyzed on the intact level. CZE-MS enabled the selective separation of several size and charge variants as well as multiple other minor variants and reduced variants. IEX-MS and CIEF-MS enabled the separation and detection of several size and charge variants as well as the detection of some other high abundant minor mass changes of the antibody. Even though CIEF and IEX using online ultraviolet (UV) detection proved to selectively separate multiple mAb variants both approaches need further improvement when it comes to MS coupling. Overall, the presented methods provide a high resolving power, separation of acidic and basic variants and different method selectivities. The methods were tested using a sample set of ten mAbs without any mAb-specific method optimization. This highlights the generic application of these methods within the tested pI range. The online UV detection was supplemented by detailed variant characterization and identification using mass spectrometry allowing an in-depth analysis of mAb variants.