The T-Cell redirecting biotherapeutic was successfully characterized using two distinct and orthogonal workflows on the same Orbitrap Excedion Pro BioPharma Hybrid MS instrument. Initially, the enhanced mass range facilitated effective analysis through Native SEC-MS, which allowed for a comprehensive evaluation of bispecific product quality by separating size variants via Size Exclusion Chromatography (SEC) and determining their masses through Native MS. Detailed subunit analyses were crucial for identifying the covalent bond linking the two monomers to form a dimer. Additionally, the mass of the bispecific was confirmed by MS with an accuracy of within 10 parts per million (ppm), and unknown peaks were identified. Given that multi-specific antibodies are prone to mis-paired and partial mAb impurities, the MS results were essential for confirming species such as dimer and trimer aggregates.

In a complementary approach, the same bispecific was analyzed using a peptide mapping workflow to confirm sequence, identify sequence variants, and assess post-translational modifications (PTMs). Enhancements to the MS instrument enabled faster electron transfer dissociation (ETD) reaction kinetics, facilitating a hybrid activation mode (EThcD) that operated as a single activation mode rather than requiring two separate experiments (ETD and higher-energy collisional dissociation, HCD). This presentation will explore various attributes of reaction times in relation to fragmentation completeness and peptide sequence coverage, as well as the impact of varying levels of supplemental activation energy. Furthermore, a detailed analysis of peptide charge state and size characteristics, coupled with universal ETD/EThcD settings, was performed to thoroughly evaluate this new Orbitrap MS instrument's usability for biotherapeutic characterization and its potential for new operational modes that optimize MS2 depth and impurity detection in a single fragmentation experiment. Finally, the integration of the orthogonal modes of analysis on the same platform was considered to achieve a holistic assessment of the biotherapeutic.

Session Speaker:

Andrew Mahan, Johnson & Johnson

Andrew Mahan

Appearances