A platform for characterizing therapeutic monoclonal antibody breakdown products by 2D chromatography and top-down...

With the growing commercialization of therapeutic monoclonal antibodies developed for the treatment of various diseases comes the need for increased analytical scrutiny of the impurity components contained within such drug products. Traditionally, relatively low performance and throughput analytical techniques were employed for elucidating the product-related breakdown components derived from the original molecule, including N-terminal Edman sequencing and matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry. Although N-terminal sequencing provides a definitive starting point of an unknown breakdown product, the resolution and mass accuracy of MALDI-TOF instruments are often insufficient for unambiguous sequence characterization. Described here is the implementation of existing advanced analytical technologies, including high-performance mass spectrometry (LTQ-Orbitrap XL-ETD) and a chip-based nanoelectrospray autosampling robot (TriVersa NanoMate), for the thorough identification and characterization of breakdown products derived from a force-degraded monoclonal antibody. Many anticipated breakdown products were identified, including Fab fragment (48,325 Da) and heavy chain polypeptide hydrolysis product (15,521 Da). Using high-resolution collisionally induced and electron transfer dissociation methods, additional identifications were made with specific localization of unpredicted modifications. As examples, a modified Fab fragment (N- and C-terminal cyclization, 47,902 Da) and a hydrolyzed free light chain impurity components (23,191 Da) were identified with a high degree of confidence (E value, <1e-5). This work describes the approach for top-down characterization of breakdown products and is readily applicable to additional monoclonal antibodies (mAb) characterization experiments, including charge isoform characterization and aggregate analysis, for a more thorough understanding of therapeutic mAb drug products.