Session: Parallel session 9 - Top-down and Structural analysis

Characterization and relative quantification of oxidation in monoclonal antibodies by a multi-level approach based on mass spectrometry.

Monoclonal antibodies (mAbs) are considered as the most rapidly growing class of therapeutics to treat several diseases. Critical quality attributes associated to mAbs such as sequence assessment or charge variants have to be monitored for safety concerns. While classical bottom-up has proven to be a valuable asset to characterize post-translational modifications in mAbs, the cross-talk between different modifications occurring in mAbs is missing. In this context we propose a combination of middle-down (MD) and native mass spectrometry (nMs) approaches to improve the characterization and assess the proportion of mAbs oxidation products.



Oxidative stress conditions were optimized (time and concentration) and applied to two different mAbs. Oxidative species were monitored using a combination of middle-up approach, to determine the oxidized subunits, and middle-down-electron-activated-dissociation-mass spectrometry (MD-EAD-MS) to put in evidence the specific residues that were oxidized at each kinetic point, allowing to simultaneously link oxidation stoichiometry with different proteoforms. Additionally, a relative quantification method based on specific fragment ions was developed to determine the oxidation propensity of each individual residue. This information was correlated to the oxidation level of the mAbs at the subunit level showing a good correlation between both methods, thus highlighting the suitability of MD workflows to relatively quantify the positional isomers within a specific protein population. Cation-exchange chromatography coupled to nMS furthermore confirmed those trends in oxidation kinetics, as well as helped deciphering the impact of oxidation on the higher order structures (HOS).



Altogether, this multi-level approach allowed to tackle the location of oxidation sites with improved oxidative proteoform characterization, provided consistent quantification results and assessed the impact of oxidation on HOS with complementary structural information.