|Univ. Prof. Mag. Dr. Christian Huber
|DI. Dr. Therese Wohlschlager
|Top- to-bottom-characterization of monoclonal antibody variants
The topic of the dissertation is the top- to bottom-characterization of monoclonal antibody (mAb) variants utilizing high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS). The focus will lie on the oxidation of methionine residues. This modification is generated by reactive oxygen due to wrong storage conditions and is a major shelf-life-limiting factor of biopharmaceuticals, as oxidized mAb variants may differ in bioactivity as well as pharmacokinetics. Therefore the development of analytical methods to monitor antibody variants is of high relevance.
In order to establish methods for characterization of mAb oxidation variants, the therapeutic antibody Rituximab will be subjected to chemical oxidation by H2O2 in order to obtain samples of defined composition. Oxidized mAb samples will be analyzed on the intact protein level, after limited proteolysis and by peptide mapping, performing capillary ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), hyphenated to a quadrupole-Orbitrap mass spectrometer.
Based on these results, absolute quantification of oxidation by means of UV-spectroscopy as well as full scan MS, will be performed.