Capillary electrophoresis for modern biopharmaceuticals: mastering large molecules and small details

The purpose of this thesis is to investigate the utility of capillary electrophoresis (CE) as an analytical technique for assessing the critical quality attributes of novel therapeutic agents for their use as innovative drugs, such as peptides, plasmids, mRNA/lipid nano particle formulations, and recombinant adeno-associated virus (rAAV). The primary objective is to enhance CE's applicability to these modalities by developing novel approaches for these challenging therapeutics. The dissertation incorporates four projects that were covered in a total of five articles published in peer-reviewed analytical chemistry periodicals. The first chapter investigates the ability of affinity CE to quantify the degree and location of methionine oxidation in peptides and monoclonal antibodies with the aim of providing a complementary approach to mass spectrometry. The following chapter provides a direct comparison of the present analytical methodologies for the analysis of rAAV including transmission electron microscopy, analytical ultra centrifugation, capillary gel electrophoresis (CGE), capillary zone electrophoresis (CZE) and ion-pair reverse phase liquid chromatography (IP-RP-LC). The third chapter describes a method for quantifying and identifying nucleic acid therapeutics using fluorescent peptide nucleic acid (PNA) hybridization probes in CE. The final part of this work reviews various analytical methods for the analysis of high-mass oligonucleotides that are incorporated in different delivery vehicles, highlighting particularly methodological benefits and drawbacks. This includes the PNA-based CE method from the previous chapter, digital droplet PCR, CGE from the second chapter with the additional and complementary use of nuclease digestion, IP-RP-LC, and CZE which were all applied for the comprehensive characterization of oligonucleotides and their quality attributes as biologics.